Antibodies and Fc fusion protein modifications with enhanced persistence or pharmacokinetic stability in vivo and methods of use thereof

ABSTRACT

In certain embodiments, this present invention provides antibodies and Fc fusion proteins with enhanced pharmacokinetics, such as biotinylated antibodies or biotinylated Fc fusion polypeptides.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application under 35 U.S.C. § 120 ofU.S. patent application Ser. No. 12/678,494 filed Apr. 14, 2010, nowU.S. Pat. No. 10,457,719, which is a 35 U.S.C. § 371 National PhaseEntry Application of International Application No. PCT/US2008/076581filed Sep. 17, 2008, which designates the U.S., and claims the benefitof priority under 35 U.S.C. 119(e) of U.S. Provisional PatentApplication No. 60/994,428 filed Sep. 18, 2007, the contents of whichare incorporated herein by reference in their entirety.

GOVERNMENT SUPPORT

Work described herein was funded, in whole or in part, by NationalInstitutes of Health Grant Number NIH R01DK056597. The United Statesgovernment has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 16, 2010, isnamed 060636_061232USC1.txt and is 9,940 bytesbytes in size.

FIELD OF THE INVENTION

The present invention relates to enhancing the in vivo persistence andstability of antibodies and Fc fusion polypeptides by biotinylation, andmethods of uses therein.

BACKGROUND OF THE INVENTION

Antibodies have been known since before the 20th century to play animportant role in immunological protection against infectious organisms.The immune system cells that produce antibodies are B-lymphocytes. Thereare four major classes: immunoglobulin M (IgM), IgG, IgA, and IgE, butIgG is by far the most prevalent class, comprising about 90% of allantibodies in adults. Each class of antibody has a specific role inimmunity, including primary and secondary immune responses, antigeninactivation and allergic reactions. IgG is the only class of antibodythat can pass the placental barrier, thus providing protection frompathogens before the newborn's immune system develops. Antibodymolecules have two ends. One end is the antigen-specific binding portionof an antibody referred to as Fab, which is highly variable andengenders each antibody with the capacity to bind a specific molecularshape. The other end, referred to as Fc, has sequence and structuralsimilarities within a class and confers the ability to bind to receptorson immunological cells that specify the effector function of antibodies.In a perfectly operating immune system, the diverse specificities of theantigen specific receptor engenders the host with a diverse repertoireof antibodies with the ability to bind to a wide array of foreigninfectious microorganisms, the result being destruction of the microbeand gain of immunity.

Most molecules, including IgM and IgE antibodies, only remain a shortamount of time in the circulation because such proteins are constantlybeing taken up by the process of fluid phase endocytosis. Thisconstitutive biological process results in the targeting of theendocytic material through the early endosomal compartment to thelysosomes, where the material is efficiently destroyed by a processreferred to as catabolism (reviewed in (Waldmann and Strober, 1969)). Ithas been established that antibodies of the IgG class have a greatlyextended half-life in circulation. This increase is the direct result ofa unique Fc receptor for IgG molecules, the neonatal Fc receptor orFcRn, which is also known as Fcgrt or FcRp (reviewed in (Ghetie andWard, 2000, 2002; Roopenian and Akilesh, 2007)). FcRn greatly slows thecatabolism of the IgG molecules by binding them in the acidic earlyendosomal cellular compartment before they enter the lysosomaldegradation pathway, causing instead the recycling of the IgG antibodiesback to the cell surface where they are released in the neutralextracellular pH environment into the circulation (reviewed in (Ghetieand Ward, 2000, 2002; Roopenian and Akilesh, 2007)). The net effect is asubstantial increase in the half-life of IgG antibodies in circulationcompared with those of proteins that lack the Fc region and are notrescued and recycled by the FcRn mediated pathway. Several investigatorshave indirectly demonstrated such a protective effect by coupling the Fcregion of IgG to different polypeptides to improve stability of thepolypeptides. In addition, the use of immunoglobulin-like domains inincreasing the stability and longevity of pharmaceutical compositionsfor therapeutic and diagnostic purposes has also been suggested (U.S.Pat. No. 6,277,375).

A key element in drug development is to achieve adequate circulatinghalf-lives, which impact dosing, drug administration and efficacy. Manyapproaches have been undertaken with the aim to increase the half-lifeof biotherapeutics. Small proteins below 60 kD are cleared rapidly bythe kidney and therefore do not reach their target. This means that highdoses are needed to reach efficacy. The modifications currently used toincrease the half-life of proteins in circulation include: PEGylation;conjugation or genetic fusion with proteins, e.g., transferrin(WO06096515A2), albumin, growth hormone (US2003104578AA); conjugationwith cellulose (Levy and Shoseyov, 2002); conjugation or fusion with Fcfragments; glycosylation and mutagenesis approaches (Carter, 2006).

In the case of PEGylation, polyethylene glycol (PEG) is conjugated tothe protein, which can be for example a plasma protein, antibody orantibody fragment. The first studies regarding the effect of PEGylationof antibodies were performed in the 1980s. The conjugation can be doneeither enzymatically or chemically and is well established in the art(Chapman, 2002; Veronese and Pasut, 2005). With PEGylation the totalsize can be increased, which reduces the chance of renal filtration.PEGylation further protects from proteolytic degradation and slows theclearance from the blood. Further, it has been reported that PEGylationcan reduce immunogenicity and increase solubility. The improvedpharmacokinetics by the addition of PEG is due to several differentmechanisms: increase in size of the molecule, protection fromproteolysis, reduced antigenicity, and the masking of specific sequencesfrom cellular receptors. In the case of antibody fragments (Fab), a20-fold increase in plasma half-life has been achieved by PEGylation(Chapman, 2002).

To date there are several approved PEGylated drugs, e.g., PEG-interferonalpha2b (PEG-INTRON) marketed in 2000 and alpha2a (Pegasys) marketed in2002. A PEGylated antibody fragment against TNF alpha, called Cimzia orCertolizumab Pegol, was filed for FDA approval for the treatment ofCrohn's disease in 2007 and has been approved on Apr. 22, 2008. Alimitation of PEGylation is the difficulty in synthesizing longmonodisperse species, especially when PEG chains over 1000 kD areneeded. For many applications, polydisperse PEG with a chain length over10000 kD is used, resulting in a population of conjugates havingdifferent length PEG chains, which need extensive analytics to ensureequivalent batches between productions. The different length of the PEGchains may result in different biological activities and thereforedifferent pharmacokinetics. Another limitation of PEGylation is adecrease in affinity or activity as it has been observed withalpha-interferon Pegasys, which has only 7% of the antiviral activity ofthe native protein, but has improved pharmacokinetics due to theenhanced plasma half-life.

Another approach is to conjugate the drug with a long lived protein,e.g. albumin, which is 67 kD and has plasma half-life of 19 days inhuman (Dennis et al., 2002). Albumin is the most abundant protein inplasma and is involved in plasma pH regulation, but also serves as acarrier of substances in plasma. In the case of CD4, increased plasmahalf-life has been achieved after fusing it to human serum albumin (Yehet al., 1992). Other examples for fusion proteins are insulin, humangrowth hormone, transferrin and cytokines (Ali et al., 1999; Duttaroy etal., 2005; Melder et al., 2005; Osborn et al., 2002a; Osborn et al.,2002b; Sung et al., 2003) and see (US2003104578A1, WO06096515A2, andWO07047504A2, herein incorporated in entirety by reference).

The effect of glycosylation on plasma half-life and protein activity hasalso been extensively studied. In the case of tissue plasminogenactivator (tPA) the addition of new glycosylation sites decreased theplasma clearance, and improved the potency (Keyt et al., 1994).Glycoengineering has been successfully applied for a number ofrecombinant proteins and immunoglobulins (Elliott et al., 2003; Raju andScallon, 2007; Sinclair and Elliott, 2005; Umana et al., 1999). Further,glycosylation influences the stability of immunoglobulins (Mimura etal., 2000; Raju and Scallon, 2006).

Another molecule used for fusion proteins is the Fc fragment of an IgG(Ashkenazi and Chamow, 1997). The Fc fusion approach has been utilized,for example in the Trap Technology developed by Regeneron (e.g. IL1 trapand VEGF trap). The use of albumin to extend the half-life of peptideshas been described in US2004001827A1. Positive effects of albumin havealso been reported for Fab fragments and scFv-HSA fusion protein (Smithet al., 2001). It has been demonstrated that the prolonged serumhalf-life of albumin is due to a recycling process mediated by the FcRn(Anderson et al., 2006; Chaudhury et al., 2003; Smith et al., 2001).

Another strategy is to use directed mutagenesis techniques targeting theinteraction of immunoglobulins to their receptor to improve bindingproperties, i.e. affinity maturation in the Fc region. With an increasedaffinity to FcRn a prolonged half-life can be achieved in vivo (Ghetieet al., 1997; Hinton et al., 2006; Jain et al., 2007; Petkova et al.,2006a; Vaccaro et al., 2005). However, affinity maturation strategiesrequire several rounds of mutagenesis and testing. This takes time, iscostly and is limited by the number of amino acids that when mutatedresult in prolonged half-lives. Therefore, simple alternative approachesare needed to improve the in vivo half-life of biotherapeutics.Therapeutics with extended half-lifes in vivo are especially importantfor the treatment of chronic diseases, autoimmune disorders,inflammatory, metabolic, infectious, and eye diseases, and cancer,especially when therapy is required over a long time period.Accordingly, a need still exists for the development of therapeuticagents (e.g., antibodies and Fc fusion proteins) with enhancedpersistence and half-lives in circulation, in order to reduce the dosageand/or the frequency of injections of a variety of therapeutic agents.

SUMMARY OF THE INVENTION

The present invention provides, in part, novel treatments and methods ofuse therein by increasing the half-life of circulating antibodies and Fcfusion polypeptides. The invention is based upon the novel finding thatbiotinylation of the Fc domain of an antibody or a fusion polypeptidecomprising an Fc domain increases the serum half-life of thebiotinylated antibody or fusion polypeptide.

Accordingly, the invention provides a method of treating a disorder in asubject in need thereof, comprising administering to the subject aneffective amount of an antibody preparation comprising an antibodymolecule conjugated to a biotin moiety.

In one embodiment, the biotin moiety is conjugated to the antibodymolecule in an amount sufficient to increase the half-life of theantibody molecule in vivo, relative to the half-life of a correspondingantibody molecule that does not comprise a biotin moiety.

In one embodiment, the antibody preparation comprises a monoclonalantibody preparation.

In another embodiment, the antibody preparation comprises a polyclonalantibody preparation. In a further embodiment, the polyclonal antibodypreparation consists essentially of a polyclonal population ofantibodies specific for a single target molecule. In another furtherembodiment, the polyclonal antibody preparation comprises a polyclonalpopulation of antibodies specific for a plurality of differentmolecules. In a further embodiment, the polyclonal antibody preparationis a pooled preparation isolated from a plurality of humans.

In one embodiment, the antibody molecule is a human antibody, humanizedantibody, primatized antibody, chimeric antibody, antigen-bindingprotein comprising a Fc domain, antibody linked to another functionalmoiety, radiolabeled antibody, a chemolabeled antibody, fusion proteincomprising an antibody, Fc domain comprising fusion polypeptide, orfragment thereof. In one embodiment, the antibody molecule is a humanimmunoglobulin G (IgG) molecule. In a further embodiment, the antibodymolecule is selected from the group consisting of IgG1, IgG2, IgG3, andIgG4.

In one embodiment, the antibody molecule comprises a human Fc domainthat is bound by the human FcRn receptor. In one embodiment, theantibody molecule is not an antibody that binds the human FcRn receptoras an antigen.

In one embodiment, the antibody comprises a human Fc domain, and thebiotin moiety is covalently conjugated to the human Fc domain. In oneembodiment, the biotin moiety is covalently conjugated to theCH2-CH3-hinge region, and/or hinge region of the human Fc domain. In afurther embodiment, the biotin moiety is covalently conjugated to anamino acid residue. In a further embodiment, the amino acid residue is alysine. In a preferred embodiment, the biotin moiety is covalentlyconjugated to a solvent exposed lysine within the human Fc domainsequence FNWYVDGVEVHNAKTKPR (SEQ ID NO:1), FKWYVDGVEVHNAKTKPR (SEQ IDNO:2), or VSNKALPAPIEK (SEQ ID NO:3). In one embodiment, the amino acidresidue is a cysteine. In a preferred embodiment, the biotin moiety iscovalently conjugated to a cysteine residue that has been geneticallyengineered onto the Fc domain. In one embodiment, the amount of biotinmoiety conjugation to the antibody is at a ratio of at least 2 biotinmoiety molecules per antibody molecule.

In one embodiment, the subject is a mammal. In a preferred embodiment,the subject is a human.

In one embodiment, the disorder is a cancer, an inflammatory disease, aninfectious disease, a neurodegenerative disease, a metabolic disease, anautoimmune disease, or an immunodeficiency. In a preferred embodiment,the disorder can be treated by antibody therapy. In a preferredembodiment, the disorder can be treated using IVIg therapy. In oneembodiment, the disorder requires an allogeneic bone marrow transplantor a transplant. In one further embodiment, the cancer is leukemia,lymphoma, prostate cancer, melanoma, breast cancer, ovarian cancer, headand neck cancer, or colon cancer. In one further embodiment, theautoimmune disease is rheumatoid arthritis, psoriasis, allergy,Kawasaki's disease, idiopathic thrombocytopenic purpura, multiplesclerosis, Guillain-Barre syndrome, Systemic Lupus Erythematosus,myasthenia gravis, or pemphigus. In one further embodiment, theimmunodeficiency is hypogammaglobulinemia. In one further embodiment,the inflammatory disease is inflammatory bowel disease, chronicobstructive pulmonary disease, atherosclerosis or osteoarthritis.

In one embodiment, the disorder is caused by a toxin. In one furtherembodiment, the toxin is a botulism toxin or a snake venom toxin. In oneembodiment, the disorder is caused by exposure to a virus. In onefurther embodiment, the virus is a Hepatitis A virus, Hepatitis B virus,Variola virus, Rabies virus, Ebola virus, herpes simplex virus,varicella zoster virus, Epstein-Barr virus, tick-borne encephalitis, ora cytomegalovirus. In one embodiment, the disorder is caused by exposureto a bacterium. In one further embodiment, the bacterium is Bacillusanthracis.

Accordingly, the invention provides a method of treating a disorder in asubject in need thereof, the method comprising administering to thesubject an effective amount of Fc fusion polypeptide comprising a humanFc domain fused to a heterologous fusion partner polypeptide, whereinsaid human Fc domain comprises a conjugated biotin moiety.

In one embodiment, the biotin moiety is conjugated to the Fc fusionpolypeptide in an amount sufficient to increase the half-life of the Fcfusion polypeptide in vivo relative to the half-life of a correspondingFc fusion polypeptide that does not comprise a biotin moiety.

In one embodiment, the human Fc domain is a human IgG Fc domain. In afurther embodiment, the human Fc domain is selected from the groupconsisting of IgG1, IgG2, IgG3, and IgG4.

In one embodiment, the biotin moiety is covalently conjugated to thehuman Fc domain of the Fc fusion polypeptide. In one embodiment, thebiotin moiety is covalently conjugated to the CH2-CH3-hinge region,and/or hinge region of the human Fc domain. In one embodiment, thebiotin moiety is covalently conjugated to an amino acid residue. In oneembodiment, the amino acid residue is a lysine. In a preferredembodiment, the biotin moiety is covalently conjugated to a solventexposed lysine within the human Fc domain sequence FNWYVDGVEVHNAKTKPR(SEQ ID NO: 1), FKWYVDGVEVHNAKTKPR (SEQ ID NO: 2), or VSNKALPAPIEK (SEQID NO: 3). In one embodiment, the amino acid residue is a cysteine. In apreferred embodiment, the biotin moiety is covalently conjugated to acysteine residue that has been genetically engineered onto the Fcdomain. In one embodiment, the amount of biotin moiety conjugation tothe Fc fusion polypeptide is at a ratio of at least 1 biotin moietymolecule per Fc monomer of the Fc fusion polypeptide.

In one embodiment, the disorder is a cancer, an inflammatory disease, ametabolic disease, an autoimmune disease, an immunodeficiency, aneurodegenerative disease, or an eye disorder. In one embodiment, thesubject is a mammal. In one preferred embodiment, the subject is ahuman.

Accordingly, in another aspect, the invention provides an Fcdomain-containing fusion polypeptide, wherein said Fc domain comprises aconjugated biotin moiety, and wherein said Fc domain-containing fusionpolypeptide has an increased half-life in vivo relative to the half-lifeof a corresponding Fc domain-containing fusion polypeptide that does notcomprise a biotin moiety.

In one embodiment, the Fc domain comprises a human Fc domain. In oneembodiment, the Fc domain consists essentially of a human Fc domain. Inone embodiment, the Fc domain consists of a human Fc domain.

In one embodiment, the human Fc domain of the Fc domain-containingfusion polypeptide is a human IgG Fc domain. In a further embodiment,the human Fc domain is selected from the group consisting of the humanFc domains of IgG1, IgG2, IgG3, and IgG4.

In one embodiment, the biotin moiety is covalently conjugated to thehuman Fc domain of the Fc fusion polypeptide. In one embodiment, thebiotin moiety is covalently conjugated to the CH2-CH3-hinge region,and/or hinge region of the human Fc domain of the Fc fusion polypeptide.In one embodiment, the biotin moiety is covalently conjugated to anamino acid residue in the human Fc domain of the Fc fusion polypeptide.In a further embodiment, the biotin moiety is covalently conjugated to alysine. In a preferred embodiment, the biotin moiety is covalentlyconjugated to a solvent exposed lysine within the human Fc domainsequence FNWYVDGVEVHNAKTKPR (SEQ ID NO: 1), FKWYVDGVEVHNAKTKPR (SEQ IDNO: 2), or VSNKALPAPIEK (SEQ ID NO: 3) of the Fc fusion polypeptide. Inone embodiment, the amino acid residue is a cysteine. In a preferredembodiment, the biotin moiety is covalently conjugated to a cysteineresidue that has been genetically engineered onto the Fc domain.

In one embodiment, the amount of biotin moiety conjugation to theantibody is at a ratio of at least 1 biotin moiety molecule per Fcdomain monomer.

Accordingly, one aspect of the invention provides an antibodypreparation, comprising antibodies specific for a plurality of antigens,said antibodies comprising conjugated biotin, and wherein saidantibodies are isolated from a human donor.

In one embodiment, the antibodies comprising conjugated biotin haveincreased serum half-life relative to antibodies of a preparationlacking said biotin. In certain embodiments, the antibodies comprisingconjugated biotin have improved pharmacokinetics relative to antibodiesof a preparation lacking said biotin. In one embodiment, the antibodiesare pooled from a group of human individuals. In certain embodiments,the human individuals are immunized against or immune to a specificantigen. In certain embodiments, the antibody preparation comprises apharmaceutically acceptable carrier.

Accordingly, one aspect of the invention provides methods for thedelivery of a therapeutic antibody or fusion polypeptide across anepithelial layer, the method comprising administering to a subject inneed thereof, an antibody or fusion polypeptide comprising an Fc domain,wherein the Fc domain comprises conjugated biotin, and wherein thebiotinylated antibody or fusion polypeptide is delivered across theepithelial barrier with increased effectiveness relative to an antibodyor fusion polypeptide lacking biotinylation of the Fc domain.

In certain specific embodiments, the biotinylated antibodies and Fcfusion polypeptides of the present invention more efficiently traverseepithelial layers. In a further embodiment, the epithelial layersinclude the respiratory, intestinal, corneal, or olfactory epitheliallayers. In another specific embodiment, the epithelial layer is anendothelial layer. In a more specific embodiment, the endothelial layeris a blood vessel or lymphatic vessel endothelial layer.

Accordingly, in one embodiment, a method is provided for the delivery ofa therapeutic antibody or fusion polypeptide across a feto-maternalorgan, the method comprising administering to a subject in need thereof,an antibody or fusion polypeptide comprising an Fc domain, wherein theFc domain comprises conjugated biotin, and wherein the biotinylatedantibody or fusion polypeptide is delivered across the feto-maternalorgan with increased effectiveness relative to an antibody or fusionpolypeptide lacking biotinylation of the Fc domain. In a specificembodiment, the feto-maternal organ is the placenta.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows determination of the plasma half-life for biotinylatedhuman (h)IgG (HuIgG-biotin) compared to unmodified human IgG (HuIgG)with the biotinylated hIgG being 6.1 days versus 3.7 days for theunmodified hIgG. The half-life of biotinylated hIgG and unmodified hIgGwas determined in C57BL/6 mFcRn−/−hFcRn+/Tg Line 276 transgenic miceexpressing the human FcRn receptor while lacking the mouse FcRnreceptor. The filled circles are the data points from treatment withbiotinylated hIgG, and the open circles from unmodified hIgG.

FIG. 2 shows determination of the plasma half-life for biotinylated hIgGcompared to unmodified hIgG. The C57BL/6 mFcRn−/−hFcRn+/Tg Line 276transgenic mouse model expressing the human FcRn receptor while lackingthe mouse FcRn receptor was used for the in vivo study. HIgG-biotin (AP)is the data obtained when biotinylated hIgG was assayed using mouseanti-human kappa-alkaline phosphatase (AP); hIgG-biotin (HRP) is thedata obtained when biotinylated hIgG was assayed usingstreptavidin-horse radish peroxidase (HRP). hIgG is the non-biotinylatedhIgG control. Both methods, AP and HRP, give comparable results withbiotinylation the half-life is extended to 6 days versus 3 days forunmodified hIgG.

FIG. 3 shows determination of the plasma half-life of hIgG dependent ofbiotin:hIgG ratio. The C57BL/6 mFcRn−/−hFcRn+/Tg Line 276 transgenicmouse model expressing the human FcRn receptor while lacking the mouseFcRn receptor was used for the in vivo study. The figure shows that onaverage two biotin molecules per IgG is needed to increase half-life andthat half-life is increased with additional biotin molecules per IgGmolecule.

FIG. 4 shows determination of the plasma half-life biotinylated hIgG-Fcfragment (hIgG-Fc-biotin) compared to the unmodified Fc fragment (hIgG).The C57BL/6 mFcRn−/−hFcRn+/Tg Line 276 transgenic mouse model expressingthe human FcRn receptor while lacking the mouse FcRn receptor was usedfor the in vivo study. This figures shows that biotin conjugated to theFc fragment can increase the half-life of such by more than 2-fold.

FIG. 5 shows that extension of plasma half-life is dependent on the FcRnreceptor. Biotinylated hIgG and unmodified hIgG was injected intoC57BL/6J FcRn−/−mice and the plasma half-life was determined. Nodifference between biotinylated hIgG and unmodified hIgG was detected.

FIG. 6 shows analysis of plasma half-life of human serum albumin (HSA)to biotinylated human serum albumin (HSA-Biotin). The C57BL/6mFcRn−/−hFcRn+/Tg Line 276 transgenic mouse model was used for the invivo study. Biotinylation did not alter the half-life of human serumalbumin.

FIG. 7 depicts hIgG conjugated to biotin using NETS-biotin (NHS-biotin)or NHS-LC-biotin (LC-biotin). Further, the structure for C6-succinimidyl4-formylbenzoate (C6-SFB) is shown, which is described in Example 7.

FIG. 8 shows plasma half-life analysis of different biotin spacers. TheC57BL/6 mFcRn−/−hFcRn+/Tg Line 276 transgenic mouse model was used forthe in vivo study. HIgG was biotinylated with NHS-biotin (HuIgG-biotin;open squares) or with NHS-LC biotin (HuIgG-LC-biotin; filled triangles)or not biotinylated (HuIgG; filled circles) and the half-life wasdetermined. Both NHS-biotinylation moieties improved the half-life ofhIgG.

FIG. 9 shows half-life analysis of hIgG1 with, and without biotinconjugation. The C57BL/6 mFcRn−/−hFcRn+/Tg Line 276 transgenic mousemodel expressing the human FcRn receptor while lacking the mouse FcRnreceptor was used for the in vivo study. HIgG was biotinylated withNHS-biotin (hIgG1-biotin; filled circles) or not biotinylated (hIgG1;open circles) and the half-life was determined. Biotinylation increasedthe half-life by 2.2-fold. In panel A the effect of biotinylated hIgG1is analyzed in C57BL/6 mFcRn−/−hFcRn+/Tg Line 276 transgenic micecarrying one copy of the human FcRn transgene and in panel B with twocopies of the human FcRn transgene similarly showing that biotinylationof hIgG1 yields more than a two-fold increase in half-life in vivo.

FIG. 10 shows half-life determination of mouse IgG1 (mIgG1) with (filledtriangles or circles) and without (open triangles or circles) biotinconjugation in C57BL/6J mice (triangles), and C57BL/6J mice lacking FcRn(circles). Biotinylation did not affect the half-life of mouse IgG1.

FIG. 11 shows that the biotin analog iminobiotin can extend thehalf-life of chimeric IgG1. The C57BL/6 mFcRn−/−hFcRn+/Tg Line 276transgenic mouse model expressing the human FcRn receptor while lackingthe mouse FcRn receptor was used for the in vivo study. The chimericantibody HuLys11 was biotinylated with NHS-biotin (HuLys11-biotin;circles), with iminobiotin (HuLys11-imminobiotin, triangles) or notbiotinylated (HuLys11; squares) and the half-life was determined.Biotinylation increased the half-life by 1.86-fold for HuLys11-biotinand 1.48-fold for HuLys11-imminobiotin compared to the unbiotinylatedHuLys11 antibody.

FIG. 12 shows biotin as a functional molecule. Human IgG (hIgG) wasconjugated to biotin using NHS-biotin (biotin) or to C6-succinimidyl4-formylbenzoate (C6-SFB) without biotin, as a similarly sized spacermoiety. The C57BL/6 mFcRn−/−hFcRn+/Tg Line 276 transgenic mouse modelexpressing the human FcRn receptor while lacking the mouse FcRn receptorwas used for the in vivo study. HIgG was derivatized with NHS-biotin(HuIgG-biotin; open squares and filled squares) or with C6-SFB(HuIgG-SF-6; open circles) or not biotinylated (HuIgG; filled circles)and the half-life was determined. Two separately derivatized biotinpreparations were used (042506 and 051304). Only biotinylation improvedthe half-life, about 1.4-fold compared with untreated hIgG and C6-SFBtreated hIgG.

FIG. 13 shows that IVIg protects mice from ankle inflammation induced byserum from a rheumatoid arthritis patient. Groups of 3Fcgr2b−/−mFcRn−/−hFcRn Tg Line 32 transgenic mice were injected on days1, 3, 8 with 0.5 ml of arthritic serum and 1 G/Kg IVIg (IVIg) or humanserum albumin (Alb) and monitored for ankle swelling and overallinflammation. IVIg treatment but not treatment with equivalent doses ofhuman serum albumin resulted in the amelioration ankle swelling andoverall inflammation.

FIG. 14 shows the effect of biotinylation on serum half-life of the Fcfragment of the humanized monoclonal antibody Herceptin (trastuzumab)using the C57BL/6 mFcRn−/−hFcRn+/Tg Line 276 transgenic mouse model. Thebiotinylated purified human Fc fragment of Herceptin Hu4D5-IgG1 (filledcircles) was analyzed in comparison to unbiotinylated antibodies (opencircles) in the transgenic mouse model. In both cases, biotinylationresults in an at least two-fold increase of half-life in vivo.

FIG. 15 shows results from a competitive binding assay in vitroinvestigating avidity of biotinylated and non-biotinylated hIgG to humanFcRn.

DETAILED DESCRIPTION OF THE INVENTION

Certain aspects of the present invention are based, at least in part, onthe discovery that biotinylation of an antibody, immunoglobulin, or Fcfusion polypeptide significantly extends its serum half-life relative tothe non-biotinylated antibody, immunoglobulin, or Fc fusion polypeptide.Although not wishing to be bound by any particular mechanism or theory,it is believed that the enhanced stability of the biotinylated antibody,immunoglobulin, or Fc fusion polypeptide is dependent on expression ofthe MHC class I-related receptor FcRn (also referred to as FcRp orFcgrt). Furthermore, it is known that the human FcRn is very stringentregarding its specificity and binds human Fc, but not mouse, rat,bovine, or sheep Fc. The FcRn can bind to two sites of the IgG (Sanchezet al., 1999; Schuck et al., 1999; West A. P. and Bjorkman, 2000). MouseIgGs do not bind efficiently to human FcRn and therefore have a shorthalf life in humans (Frodin et al., 1990). In contrast, mouse FcRn bindsIgG from every species analyzed (Ober et al., 2001).

Most serum proteins have a short serum half-life (about 1-2 days).However, two types of serum proteins, albumin and antibodies of the IgGclass, have greatly extended serum half-lives. For example, mostsubclasses of IgG have a half-life of about 10-20 days in humans. The Fcregion of IgG is required for this extension of half-life. Thus,truncated IgG polypeptides carrying only the Fc region also show suchextended serum half-life. Moreover, when the Fc region is fused with afusion partner (e.g., a biologically active protein), this Fc fusionprotein shows an extended serum half-life due to its interaction withFcRn. The mechanism by which FcRn extends the serum half-life of IgG andIgG Fc fusion proteins is well established (Ghetie and Ward, 2000, 2002;Roopenian and Akilesh, 2007). FcRn is localized in the endosomalcompartments of many cell types, including vascular endothelium. Serumproteins are constantly being endocytosed and directed to the earlyendosomal vesicles. FcRn is harbored primarily in this acidifiedvesicle. In this acidified environment, the Fc region binds FcRn, andthe IgG/FcRn complex is then recycled either apically or basolaterallyback to the plasma membrane, whereupon exposure to the neutral pH 7.2extracellular environment results in its release into the circulation.In contrast, other endocytosed proteins that do not bind FcRn are notrescued, and thus continue though the endosomal route to catabolicelimination, resulting in their short half-life. The biochemicalmechanism by which the Fc region of IgG binds FcRn in an acidicenvironment is well understood. The CH2-CH3-hinge region of the Fcregion contains solvent exposed histidine residues, which whenprotonated, engage residues on FcRn with sufficient affinity to permitIgG to exploit the FcRn recycling pathway to escape catabolicelimination.

Modified Antibodies

An object of the present invention is to provide methods andcompositions for treating a disorder in a subject, comprisingadministering to the subject an effective amount of an antibody moleculecomprising a biotin moiety. Accordingly in one embodiment, the biotinmoiety is conjugated to the antibody molecule in an amount sufficient toincrease the half-life of the antibody molecule in vivo relative to thehalf-life of a corresponding antibody molecule that does not comprise abiotin moiety. In a preferred embodiment, the antibody moleculecomprising a biotin moiety comprises an Fc fragment that is capable ofbinding to the human FcRn receptor. In a preferred embodiment, theantibody molecule comprising a biotin moiety is not an antibody thatspecifically recognizes the human FcRn receptor as an antigen.

As used herein, the term “antibody” refers to an intact immunoglobulinor to a monoclonal or polyclonal antigen-binding fragment with the Fc(crystallizable fragment) region or FcRn binding fragment of the Fcregion, referred to herein as the “Fc fragment” or “Fc domain”.Antigen-binding fragments may be produced by recombinant DNA techniquesor by enzymatic or chemical cleavage of intact antibodies.Antigen-binding fragments include, inter alia, Fab, Fab′, F(ab′)2, Fv,dAb, and complementarity determining region (CDR) fragments,single-chain antibodies (scFv), single domain antibodies, chimericantibodies, diabodies and polypeptides that contain at least a portionof an immunoglobulin that is sufficient to confer specific antigenbinding to the polypeptide. The Fc domain includes portions of two heavychains contributing to two or three classes of the antibody. The Fcdomain may be produced by recombinant DNA techniques or by enzymatic(e.g. papain cleavage) or via chemical cleavage of intact antibodies.

An immunoglobulin is typically a tetrameric molecule. As used herein,the term “immunoglobulin” refers to one or more chains of the tetramericmolecule. In a naturally occurring immunoglobulin, each tetramer iscomposed of two identical pairs of polypeptide chains, each pair havingone “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa). Theamino-terminal portion of each chain includes a variable region of about100 to 110 or more amino acids primarily responsible for antigenrecognition. The carboxy-terminal portion of each chain defines aconstant region primarily responsible for effector function. Human lightchains are classified as kappa and lambda light chains. Heavy chains areclassified as mu, delta, gamma, alpha, or epsilon, and define theantibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Withinlight and heavy chains, the variable and constant regions are joined bya “J” region of about 12 or more amino acids, with the heavy chain alsoincluding a “D” region of about 10 more amino acids. See generally,Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y.(1989)) (incorporated by reference in its entirety for all purposes). Inhuman, there are in addition four IgG (IgG1, IgG2, IgG3 and IgG4) andtwo IgA subtypes present. The classification is done according todifferences in their heavy chain constant domains (for review seeJaneway C A, Jr. et al (2001), Immunobiology, 5th ed.; Pier G B, LyczakJ B, Wetzler L M (2004), Immunology, Infection, and Immunity, ASM PressISBN 1-55581-246-5, both herein incorporated in entirety by reference).The variable regions of each light/heavy chain pair form the antibodybinding site such that an intact natural immunoglobulin has two bindingsites.

Immunoglobulin chains exhibit the same general structure: they includerelatively conserved framework regions (FR) joined by threehypervariable regions, also called complementarity determining regionsor CDRs. The CDRs from the two chains of each pair are aligned by theframework regions, enabling binding to a specific epitope. FromN-terminus to C-terminus, both light and heavy chains comprise thedomains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of aminoacids to each domain is in accordance with the definitions of KabatSequences of Proteins of Immunological Interest (National Institutes ofHealth, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk J. Mol. Biol.,1997, 196:901-917; Chothia et al. Nature, 1989, 342:878-883 (1989).

The antibody comprising a biotin moiety may be an IgG, an IgM, an IgE,an IgA or an IgD molecule. In a preferred embodiment, the antibodymolecule comprising a biotin moiety is an IgG, and more preferably is ofthe IgG1, IgG2, IgG3, or IgG4 subtype. The class and subclass ofantibodies may be determined by any method known in the art, forexample, by using antibodies that are specific for a particular classand subclass of antibody. Such antibodies are available commercially.The class and subclass can be determined by ELISA and Western Blots, aswell as other techniques. Alternatively, the class and subclass may bedetermined by sequencing all or a portion of the constant domains of theheavy and/or light chains of the antibodies, comparing their amino acidsequences to the known amino acid sequences of various class andsubclasses of immunoglobulins, and determining the class and subclass ofthe antibodies.

Antibodies are a major class of biopharmaceuticals. Antibodies fortherapeutic purposes are often produced from a cell line (e.g. CHOcells, the hamster line BHK21, the human PER.C6 cell line, COS, NIH 3T3,BHK, HEK, 293, L929, MEL, JEG-3, murine lymphoid cells (including NS0and Sp2/0-Ag 14)), including hybridomas, and are usually a single cloneof a specific antibody. Antibodies used for therapeutic purposes areclassified as murine, chimeric, humanized or fully human antibodies andare produced by recombinant methods. A “murine antibody” is a full mouseantibody and has only limited use in humane due to its short half-lifein circulation and its high immunogenicity. A “chimeric antibody” is agenetically engineered antibody, which contains both mouse and humansequences. The ratio is approximately 33% mouse contribution and 67%human contribution. Usually the variable domains are murine and theconstant region including the Fc fragment is derived from a human IgG.

A “humanized antibody” is a genetically engineered antibody, wherein themouse content is reduced to about 5-10%. In such cases, the six CDRs ofthe heavy and light chains and a limited number of structural aminoacids of the murine monoclonal antibody are grafted by recombinanttechnology to the CDR-depleted human IgG scaffold. A fully humanantibody or human antibody describes antibodies, which are made inhumanized mice resulting in antibodies that do not contain any mousesequences, or made in vitro using phage libraries or ribosome display oralternatively are obtained from human donors. In certain embodiments,chimeric, humanized or primatized (CDR-grafted) antibodies, comprisingportions derived from different species or fully human antibodies, arealso encompassed by the present invention as target molecules to bebiotinylated. The various portions of these antibodies can be joinedtogether chemically by conventional techniques, or can be prepared as acontiguous protein using genetic engineering techniques. For example,nucleic acids encoding a chimeric or humanized chain can be expressed toproduce a contiguous protein. See, e.g., Cabilly et al., U.S. Pat. No.4,816,567; Cabilly et al., European Patent No. 0,125,023; Boss et al.,U.S. Pat. No. 4,816,397; Boss et al., European Patent No. 0,120,694;Neuberger, M. S. et al., WO 86/01533; Neuberger, M. S. et al., EuropeanPatent No. 0,194,276 B1; Winter, U.S. Pat. No. 5,225,539; and Winter,European Patent No. 0,239,400 B1. See also, Newman, R. et al.,BioTechnology, 10: 1455-1460 (1992), regarding primatized antibody. See,e.g., Ladner et al., U.S. Pat. No. 4,946,778; and Bird, R. E. et al.,Science, 242: 423-426 (1988)), regarding single chain antibodies.

In addition, functional fragments of antibodies, including fragments ofchimeric, humanized, or primatized or fully human antibodies can bebiotinylated according to the present invention. Functional fragments ofthe subject antibodies retain at least one binding and one Fc fragmentfunction of the full-length antibody from which they are derived.Preferred functional fragments retain an antigen binding function of acorresponding full-length antibody. Certain preferred functionalfragments retain the ability to inhibit one or more functions, such as abinding activity or a transport activity. In certain embodiments, theantibody may be used as a targeting agent to deliver a payload to atarget cell.

In addition, a mixture of antibodies, termed herein as an “antibodypreparation”, can be used according to the methods of the presentinvention. Such antibody preparations include polyclonal and monoclonalmixtures of antibodies. Accordingly, an object of the present inventionis to provide methods of treating a disease or disorder comprisingadministering an antibody preparation comprising conjugated biotinmoieties, wherein said biotin moiety conjugated antibodies in saidantibody preparation have increased serum half-life relative toantibodies of a preparation lacking the biotin moiety.

In one embodiment, the antibody preparation comprises polyclonalantibodies for use in intravenous immunoglobulin (IVIg) therapies. IVIghas been used since the early 1980s to treat immunological disorders.IVIg preparations usually contain more than 95 percent unmodified IgG,obtained from pooled human donors. Currently IVIg products are used totreat a wide range of disorders including immunodeficiencies, sepsis,neurological disorders, autoimmune and inflammatory diseases, and arealso beneficial for transplantation reducing the allograft rejection.

In one embodiment, the antibody preparation is in addition sialylated atthe N-linked glycan of the IgG Fc fragment, which can substantiallyenhance its anti-inflammatory effect by a mechanism distinct from FcRnbinding (Kaneko et al., Science 313(5787), 670-673 (2006); Anthony etal., Science 320(5874), 373-376 (2008)). Biotinylation of IVIg acting toextend its serum half-life and concentrations maintained in circulationmay thus further augment the efficacy of sialylated IgG and derivativesthereof.

In specific embodiments, the present invention relates to biotinylationof immunoglobulins, chimeric antibodies, humanized antibodies, or fullyhuman antibodies for use in methods of treatment. For example, ahumanized antibody can be an antibody derived from a non-human species,in which certain amino acids in the framework and constant domains ofthe heavy and light chains have been mutated so as to reduce of abolishan immune response in humans. Alternatively, a humanized antibody may beproduced by fusing the constant domains from a human antibody to thevariable domains of a non-human species. Examples of how to makehumanized antibodies may be found in U.S. Pat. Nos. 6,054,297, 5,886,152and 5,877,293.

A humanized antibody may comprise portions of immunoglobulins ofdifferent origin. For example, at least one portion can be of humanorigin. For example, the humanized antibody can comprise portionsderived from an immunoglobulin of nonhuman origin with the requisitespecificity, such as a mouse, and from immunoglobulin sequences of humanorigin (e.g., a chimeric immunoglobulin), joined together chemically byconventional techniques (e.g., synthetic) or prepared as a contiguouspolypeptide using genetic engineering techniques (e.g., DNA encoding theprotein portions of the chimeric antibody can be expressed to produce acontiguous polypeptide chain). Alternatively, a humanized antibody maybe created in a transgenic or humanized animal expressing the humanantibody genes (see Lonberg, N. “Transgenic Approaches to HumanMonoclonal Antibodies.” Handbook of Experimental Pharmacology 113(1994): 49-101).

Another example of a humanized antibody of the present invention is animmunoglobulin containing one or more immunoglobulin chains comprising aCDR of nonhuman origin (e.g., one or more CDRs derived from an antibodyof nonhuman origin) and a framework region derived from a light and/orheavy chain of human origin (e.g., CDR-grafted antibodies with orwithout framework changes). Chimeric or CDR-grafted single chainantibodies are also encompassed by the term “humanized antibody”.

Methods of preparing immunoglobulins, immunizing with antigens, andpolyclonal and monoclonal antibody production can be performed asdescribed herein, or using other suitable techniques. A variety ofmethods have been described. See e.g., Kohler et al., Nature, 256:495-497 (1975) and Eur. J. Immunol. 6: 511-519 (1976); Milstein et al.,Nature 266: 550-552 (1977); Koprowski et al., U.S. Pat. No. 4,172,124;Harlow, E. and D. Lane, 1988, Antibodies: A Laboratory Manual, (ColdSpring Harbor Laboratory: Cold Spring Harbor, N.Y.); Current ProtocolsIn Molecular Biology, Vol. 2 (Supplement 27, Summer '94), Ausubel, F. M.et al., Eds., (John Wiley & Sons: New York, N.Y.), Chapter 11, (1991;Rasmussen S K, Rasmussen L K, Weilguny D, Tolstrup A B. Biotechnol Lett.2007 Feb. 20.). Generally, a hybridoma can be produced by fusing asuitable immortal cell line (e.g., a myeloma cell line such as SP2/0)with antibody producing cells. The antibody producing cell, preferablythose of the spleen or lymph nodes, are obtained from animals immunizedwith the antigen of interest. The fused cells (hybridomas) can beisolated using selective culture conditions, and cloned by limitingdilution. Cells that produce antibodies with the desired specificity canbe selected by a suitable assay (e.g., ELISA).

Antibodies can be purified from the plasma of human donors. This is thecurrent approach for IVIg where immunoglobulins are isolated from normalplasma pooled from a large number of donors (seehttp://www.fda.gov./cber/gdlns/igivimmuno.htm and Haeney Clin ExpImmunol. 1994 July; 97(Suppl 1): 11-15.) At times IVIg is referred toherein as IGIV.

Other suitable methods for producing or isolating antibodies of therequisite specificity can be used, including, for example, methods whichselect recombinant antibodies from a library, or which rely uponimmunization of transgenic animals (e.g., mice) capable of producing afull repertoire of human antibodies. See e.g., Jakobovits et al., Proc.Natl. Acad. Sci. USA, 90: 2551-2555 (1993); Jakobovits et al., Nature,362: 255-258 (1993); Lonberg et al., U.S. Pat. No. 5,545,806; Surani etal., U.S. Pat. No. 5,545,807.

An antibody may have one or more binding sites. If there is more thanone binding site, the binding sites may be identical to one another ormay be different. For instance, a naturally occurring immunoglobulin hastwo identical binding sites, a single-chain antibody or Fab fragment hasone binding site, while a “bispecific” or “bifunctional” antibody hastwo different binding sites.

The term “human antibody” includes all antibodies that have one or morevariable and constant regions derived from human immunoglobulinsequences. In one embodiment, all of the variable and constant domainsare derived from human immunoglobulin sequences (a fully humanantibody). These antibodies may be prepared in a variety of ways, asdescribed below.

The term “chimeric antibody” refers to an antibody that contains one ormore regions from one antibody and one or more regions from one or moreother different antibodies. In one embodiment, one or more of the CDRsare derived from a specific human antibody. In a more preferredembodiment, all of the CDRs are derived from a human antibody. Inanother preferred embodiment, the CDRs from more than one human antibodyare mixed and matched in a chimeric antibody. For instance, a chimericantibody may comprise a CDR1 from the light chain of a first humanantibody combined with CDR2 and CDR3 from the light chain of a secondhuman antibody, and the CDRs from the heavy chain may be derived from athird antibody. Further, the framework regions may be derived from oneof the same antibodies, from one or more different antibodies, such as ahuman antibody, or from a humanized antibody.

In certain embodiments, the antibody is linked to an additionalfunctional moiety. Such linkage may be covalent or non-covalent. In oneembodiment, the functional moiety may be therapeutic, e.g., a drugconjugate or toxin.

In certain embodiments, the antibody is in addition glycosylated orsialylated.

In certain embodiments, the antibody may be covalently linked tocytotoxic drugs, small molecules, enzymatic proteins, plant toxins,bacterial toxins, radioisotopes or radionuclides (Junutula et al., 2008;Polakis, 2005; Wu and Senter, 2005). Examples of cytotoxins arecalicheamicin, duocarmycin, mertanisien, maytansinoids, auristatin,131I, 90Y, 214Bi, Pseudomonas Exotoxin PE38, Diphteria toxin, ricin,saporin, doxorubicin and RNase.

In certain further embodiments, the antibody is labeled to facilitatedetection. As used herein, the terms “label” or “labeled” refers toincorporation of another molecule in the antibody. In one embodiment,the label is a detectable marker, e.g., incorporation of a radiolabeledamino acid. Various methods of labeling polypeptides and glycoproteinsare known in the art and may be used. Examples of labels forpolypeptides include, but are not limited to, the following:radioisotopes or radionuclides (e.g., 3H, 14C, 15N, 35S, 90Y, 99Tc,111In, 125I, 131I), fluorescent labels (e.g., FITC, rhodamine,lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase,beta-galactosidase, luciferase, alkaline phosphatase), chemiluminescentmarkers, biotinyl groups, predetermined polypeptide epitopes recognizedby a secondary reporter (e.g., leucine zipper pair sequences, bindingsites for secondary antibodies, metal binding domains, epitope tags),magnetic agents, such as gadolinium chelates, toxins such as pertussistoxin, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin,doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,procaine, tetracaine, lidocaine, propranolol, and puromycin and analogsor homologs thereof.

In certain embodiments, the antibodies are further attached to a labelthat can be detected (e.g., the label can be a radioisotope, fluorescentcompound, enzyme or enzyme co-factor). The active moiety may be aradioactive agent, such as: radioactive heavy metals such as ironchelates, radioactive chelates of gadolinium or manganese, positronemitters of oxygen, nitrogen, iron, carbon, or gallium, ⁴³K ⁵⁷Co, ⁶⁷Cu,⁶⁷Ga, ⁶⁸Ga, ¹²³I, ¹²⁵I, ¹³¹I, ¹³²I, or ⁹⁹Tc. A binding agent affixed tosuch a moiety may be used as an imaging agent and is administered in anamount effective for diagnostic use in a mammal such as a human and thelocalization and accumulation of the imaging agent is then detected. Thelocalization and accumulation of the imaging agent may be detected byradioscintigraphy, nuclear magnetic resonance imaging, computedtomography, or positron emission tomography. Immunoscintigraphy usingantibodies or other binding polypeptides may be used to detect and/ordiagnose cancers and vasculature. For example, monoclonal antibodieslabeled with ⁹⁹Technetium, ¹¹¹Indium, or ¹²⁵Iodine may be effectivelyused for such imaging. As will be evident to the skilled artisan, theamount of radioisotope to be administered is dependent upon theradioisotope. Those having ordinary skill in the art can readilyformulate the amount of the imaging agent to be administered based uponthe specific activity and energy of a given radionuclide used as theactive moiety. Typically 0.1-100 millicuries per dose of imaging agent,preferably 1-10 millicuries, and most often 2-5 millicuries areadministered. Thus, compositions according to the present inventionuseful as imaging agents comprising a targeting moiety conjugated to aradioactive moiety comprise 0.1-100 millicuries, in some embodimentspreferably 1-10 millicuries, in some embodiments preferably 2-5millicuries, in some embodiments more preferably 1-5 millicuries.

In other embodiments, the antibodies are manufactured in prokaryotic oreukaryotic cells capable of being transformed or transfected withexogenous DNA and grown in culture. Such prokaryotic cells areEscherichia coli or Bacillus. Such eukaryotic cells are mammalian orfungal cells, algae or plant cells. Such mammalian cells include Chinesehamster ovary (CHO), murine lymphoid cells (including NS0 and Sp2/0-Ag14), hybridomas, the hamster line BHK21, the human PER.C6 cell line andthe like. Fungal cells, including species of yeast (e.g., Saccharomycesspp., Schizosaccharomyces spp., Pichia pastoris), or filamentous fungi(e.g., Aspergillus spp., Neurospora spp.) may be used as host cellswithin the present invention. Algae or microalgae including Anabaenacylindrical, Aphanizomenon flos-aquae, Chlamydomonas rheinhardii,Chlorella pyrenoidosa, Chlorella vulgaris, Dunaliella salina, Euglenagracilis, Porphyridium cruentum, Scenedesmus obliquus, Spirogyra sp.,Arthrospira maxima, Spirulina platensis and Synechococcus sp. may beused as host cells within the present invention. In certain embodiments,the antibodies are manufactured in transgenic animals (e.g. mouse, rat,goat, sheep, pig, rabbit, bovine, chicken etc.) or transgenic plants.Transgenic plants including species of tobacco (e.g. Nicotiana tabacum),potato, pea, alfalfa, tomato, barley, and Maize.

As used herein, the term “increase the half-life” or simply “increasedhalf-life” indicates that the in vivo half-life of an antibody or Fcfusion polypeptide comprising a conjugated biotin is at least 10% higherthan the in vivo half-life of a comparable, control antibody or Fcfusion polypeptide not comprising a biotin. It is preferred that thehalf-life of an antibody or Fc fusion polypeptide comprising aconjugated biotin is at least 10% higher, at least 20% higher, at least30% higher, at least 40% higher, at least 50% higher, at least 60%higher, at least 70% higher, at least 80% higher, at least 90% higher,at least 1-fold higher, at least 2-fold higher, at least 5-fold higher,at least 10 fold higher, at least 100 fold higher, at least 1000-foldhigher, or more, than a control antibody or Fc fusion polypeptide notcomprising a biotin. To avoid doubt, the FcRn humanized mouse model,described herein in the Examples, can be used to determine in vivohalf-life of said antibodies or Fc fusion polypeptides described herein.

Fc Fusion Proteins

An object of the present invention is to provide methods of treating adisorder in a subject in need thereof, comprising administering to thesubject an effective amount of an Fc fusion polypeptide conjugated to abiotin moiety. Accordingly, the Fc fusion polypeptide comprises a humanFc domain fused to a heterologous fusion partner polypeptide, whereinthe human Fc domain comprises a conjugated biotin moiety. Theconjugation of the Fc fragment can be achieved by using geneticengineering techniques or chemical coupling methods well established inthe art.

The terms “Fc domain” or “Fc fragment”, interchangeably used herein,encompass native and altered forms of polypeptides derived from the Fcregion of an antibody, preferably a human antibody, that are bound byFcRn. The Fc domain normally has at least two heavy chain constantregion domains (CH2 and CH3). To avoid confusion, the natural human Fcdomain refers to the human Fc domain sequence encompassed by theteachings of Kabat, herein incorporated by reference Kabat Sequences ofProteins of Immunological Interest (National Institutes of Health,Bethesda, Md. (1987 and 1991)). Forms of such polypeptides containingthe hinge region that promotes dimerization are also included. Onesuitable Fc fragment, described in PCT applications WO05047334A1 and inWO04074455A2, is a single chain polypeptide extending from theN-terminal hinge region to the native C-terminus. It may be desirable touse altered forms of Fc fragments having improved serum half-life,altered effector functions, altered spatial orientation, and the like.The alteration of the Fc fragment can be achieved using geneticengineering techniques known in the art.

Immunoglobulin heavy chain constant region domains include CH1, CH2,CH3, and CH4 of any class of immunoglobulin heavy chain including gamma,alpha, epsilon, mu, and delta classes. A particularly preferredimmunoglobulin heavy chain constant region domain is human CH2 and CH3.DNA sequences encoding immunoglobulins may be cloned from a variety ofgenomic or cDNA libraries known in the art. The techniques for isolatingsuch DNA sequences using probe-based methods are conventional techniquesand are well known to those skilled in the art. Probes for isolatingsuch DNA sequences may be based on published DNA sequences (see, forexample, Hieter et al., Cell 22: 197-207, 1980). Alternatively, thepolymerase chain reaction (PCR) method disclosed by Mullis et al. (U.S.Pat. No. 4,683,195) and Mullis (U.S. Pat. No. 4,683,202), incorporatedherein by reference may be used. The choice of library and selection ofprobes for the isolation of such DNA sequences is within the level ofone of ordinary skill in the art.

In one embodiment, a particular polypeptide is fused C-terminally to theN-terminus of the constant region of immunoglobulins in place of thevariable region(s) thereof, however N-terminal fusions of the bindingpartner may also be constructed. Typically, such fusions retain at leastthe functionally active hinge, CH2 and CH3 domains of the constantregion of an immunoglobulin heavy chain, herein referred to as the“CH2-CH3-hinge” region. Fusion polypeptides for use in the presentinvention are also made to the C-terminus of the Fc portion of aconstant domain, or immediately N-terminal to the CH1 of the heavy chainor the corresponding region of the light chain. This can be accomplishedby constructing the appropriate DNA sequence and expressing it inrecombinant cell culture. Alternatively, there may be a spacer (alsocalled linker) included between the Fc domain and the polypeptide. Inaddition, the Fc domain may be modified to introduce functional groupssuch as lysines or cysteines, to allow or improve the conjugation of thebiotin. Such modifications can be introduced using genetic mutagenesisapproaches known in the art.

Furthermore, the Fc domain may be synthesized according to knownmethods. For example, the Fc domain may be produced by recombinant DNAtechniques or by enzymatic (e.g. papain cleavage) or chemical cleavageof intact antibodies. Host cells for use in preparing Fc domain fusionpolypeptides include prokaryotic and eukaryotic cells capable of beingtransformed or transfected with exogenous DNA and grown in culture, suchas Escherichia coli, cultured mammalian and fungal cells or plant cells.Mammalian cells include Chinese hamster ovary (CHO,), the hamster lineBHK21, the human PER.C6 cell line, COS, NIH 3T3, BHK, HEK, 293, L929,MEL, JEG-3, murine lymphoid cells (including NS0 and Sp2/0-Ag 14),hybridoma cells. Fungal cells, including species of yeast (e.g.,Saccharomyces spp., Schizosaccharomyces spp., Pichia pastoris), orfilamentous fungi (e.g., Aspergillus spp., Neurospora spp.) may be usedas host cells within the present invention. Algae including Anabaenacylindrical, Aphanizomenon flos-aquae, Chlamydomonas rheinhardii,Chlorella pyrenoidosa, Chlorella vulgaris, Dunaliella salina, Euglenagracilis, Porphyridium cruentum, Scenedesmus obliquus, Spirogyra sp.,Arthrospira maxima, Spirulina platensis and Synechococcus sp. In certainembodiments, the antibodies are manufactured in transgenic animals (e.g.goat) or transgenic plants. Transgenic plants including species oftobacco (e.g. Nicotiana tabacum), potato, pea, alfalfa, tomato, barley,Maize. Further transgenic animals maybe used to produce the recombinantprotein, for example transgenic mice, rats, chicken, goat, rabbit, pig,sheep or cow may be used.

Furthermore, the Fc domain may be in addition glycosylated or sialyated.

The polypeptide or protein fused to the biotinylated Fc domain can be agrowth factor, a cytokine, an enzyme, a receptor domain, an antibodyfragment (single chain (scFv), Fab fragment domain antibody), a camelid,a llama, an alternative binding protein, antibody substructure,minibody, adnectin, anticalin, affibody, affilin, knottin, glubody,C-type lectin-like domain protein, designed ankyrin-repeat proteins(DARPin), tetranectin, kunitz domain protein, maxybody, thioredoxin,cytochrome b562, zinc finger scaffold, Staphylococcal nuclease scaffold,fibronectin or fibronectin dimer, tenascin, N-cadherin, E-cadherin,ICAM, titin, GCSF-receptor, cytokine receptor, glycosidase inhibitor,antibiotic chromoprotein, myelin membrane adhesion molecule P0, CD8,CD4, CD2, class I MHC, T-cell antigen receptor, CD1, C2 and I-setdomains of VCAM-1, 1-set immunoglobulin domain of myosin-binding proteinC, 1-set immunoglobulin domain of myosin-binding protein H, I-setimmunoglobulin domain of telokin, NCAM, twitchin, neuroglian, growthhormone receptor, erythropoietin receptor, prolactin receptor,interferon-gamma receptor, β-galactosidase/glucuronidase,β-glucuronidase, transglutaminase, T-cell antigen receptor, superoxidedismutase, tissue factor domain, cytochrome F, green fluorescentprotein, GroEL, thaumatin, a matrix protein or the like.

Non-limiting examples of Fc fusion polypeptides are the fusion of IL-1to the Fc fragment (IL-1 trap), VEGF to the Fc fragment (VEGF trap),DNase I-Fc fusion, E-cadherin Fc fusion, PTH-Fc Fusion, single chainantibody fusion, domain antibody fusion and etanercept (human p75TNF-alpha receptor Fc fusion). The biotinylated fusion polypeptides ofthe present invention are useful in a variety of applications, includingresearch and therapeutic applications.

Biotin Moieties and Biotinylation

In certain aspects, the present invention relates to methods oftreatment involving the administration of antibodies and Fc fusionpolypeptides with biotin moieties. Biotin is also known ashexahydro-2-oxo-1H-thieno[3,4-d]imidazole-4-pentanoic acid, vitamin B7,vitamin H, Coenzyme R or Biopeiderm. Biotin was first isolated in 1936and is an essential nutrient factor in mammals. Its chemical formula isC10H16N2O3S. It is water soluble, hydrophobic and small, with amolecular weight of 244.31. Biotin is a bicyclic molecule composed of anureido ring fused with a tetrahydrothiophene (or thiophane) ring.Although biotin in doses below 5 mg daily is not toxic, when taken above5 mg daily for one month or longer dihydrotestrone-like symptoms maydevelop (Andersen, 2001). Biotin functions as a cofactor incarboxylation, decarboxylation, and transcarboxylation reactions relatedto biochemical processes, such as glucogenesis and fatty acid synthesis.

In the laboratory biotin can be covalently linked to both proteins andnucleic acids, and is widely used as a label in many molecular biologyand biochemistry technologies, especially together with avidin orstreptavidin as a detection molecule (ELISA, ELISPOT). For example,owing to the high affinity of biotin to avidin or streptavidin, biotinis commonly used as a tag for protein purification and diagnostics.Biotin has also been used as a coupling molecule for the creation ofbispecific antibodies or tetrabodies and prodrug approaches.

In certain embodiments of the present invention, biotin derivatives oranalogs can be applied. Thus, as used herein, the term “biotin moiety”includes biotin and its derivatives and analogs, such as 2-imminobiotin.Other biotin derivatives or analogs that can be applied to the objectsof the present invention include, but are not limited to, biocytin(-biotinoyl-L-lysine), biotin ethylenediamine, biotin cadaverine,biotin-X cadaverine, DSB-X desthiobiocytin (-desthiobiotinoyl-L-lysine,)and DSB-X biotin ethylenediamine and chloroacetylated biotin derivative(CABI), as well as other biotin analogs commonly known in the art. A“biotin moiety”, as the term is used herein, will increase the serumhalf-life of an antibody or Fc-domain containing fusion polypeptide.

In certain embodiments biotinylation can be achieved by a chemicalreaction in vitro or in an in vivo system, e.g. utilizing the E. colibiotin holoenzyme synthetase and is well established in the art (Bayerand Wilchek, 1990; Haugland and You, 1995, 1998; Yeo et al., 2004). Inother embodiments, through chemical conjugation, biotin can be coupledto primary and epsilon amines of lysine either directly or via a spacerto the Fc polypeptide. Here, the term “Fc polypeptide” includes Fcfragments, antibodies, antibody fragments containing an Fc fragment, aswell as Fc fusion polypeptides, or the like. The amines of the Fcpolypeptide that can be easily targeted are most commonly lysineresidues, using for example the N-hydroxysuccinimide (NHS) esterchemistry (NHS CAS Number 6066-82-6). An exemplary method is to labelthe epsilon-amino groups of lysine residues with a succinimidyl ester ofbiotin (Ugarova et al., 1979). Any primary amine or epsilon amine reactswith NHS esters and can therefore be targeted for crosslinking using theN-hydroxysuccinimide (NHS) ester chemistry.

In preferred embodiments of the present invention, the Fc fragment ofthe antibody or Fc fusion polypeptide may be mutagenized such that onlyone, two or three lysine residues are available for crosslinking to abiotin moiety. For example, lysines not to be used for biotinconjugation can be mutagenized to alanine. In a preferred embodiment,new lysine residues can be introduced to allow additional and/orsite-specific biotinylation.

Alternatively, in other embodiments, cysteines on the Fc fragment of theantibodies or Fc fusion polypeptides can be biotinylated. Specificreactive cysteine residues can be genetically engineered into the Fcfragment using methods known in the art. For example, a solvent exposedcysteinyl residue can be engineered onto IgG, such that it creates asolvent exposed cysteine, thus making it available for conjugation, forexample using Iodoacetyl-Biotin, which reacts with the sulfhydryl group(—SH) of the cysteine and forms a stabile thioether linkage. The use ofiodoacetyl compounds for linking toxins or labels to antibodies orproteins has been demonstrated. Examples include the attachment of PEGto proteins, e.g., PEGylated colony stimulating factor (pegfilgrastim),PEGylated interferon alpha (pegasys). Biotin can be conjugated to thethiol group of the cysteine using standard coupling chemistry (Wong,1991) or the THIOMAB technology as described in (Junutula et al., 2008).In one embodiment, lysine residues can be replaced by reactive cysteineresidues. As but a few examples, one or more cysteines can replace thebiotin-targeted lysine residue in the Fc domain sequence of SEQ ID NO:1,SEQ ID NO:2, and/or SEQ ID NO:3. Thus, example sequences can include:

SEQ ID NO 4: FNWYVDGVEVHNA C TKPR SEQ ID NO 5: FNWYVDGVEVHNAKT C PRSEQ ID NO 6: FNWYVDGVEVHNA C T C PR SEQ ID NO 7: F C WYVDGVEVHNAKTKPRSEQ ID NO 8: FKWYVDGVEVHNA C TKPR SEQ ID NO 9: FKWYVDGVEVHNAKT C PRSEQ ID NO 10: F C WYVDGVEVHNA C TKPR SEQ ID NO 11: F C WYVDGVEVHNAKT CPR SEQ ID NO 12: FKWYVDGVEVHNA C T C PR SEQ ID NO 13: F C WYVDGVEVHNA CT C PR SEQ ID NO 14: VSN C ALPAPIEK SEQ ID NO 15: VSNKALPAPIE CSEQ ID NO 16: VSN C ALPAPIE C

In alternative embodiments, other reactive amino acids may be targetedfor conjugation: gamma and beta-carboxyl groups of glutamic and asparticacids, imidazolyl group of histidine, thioether moiety of methionine,indolyl group of tryptophan, and phenolic hydroxyl group of tyrosine(Wong, 1991). For example, thiol or aldehyde groups of the protein maybe targets. Additionally, sulfhydryl groups of the Fc fragment can bebiotinylated using biotin iodoacetamide or maleimide. Furthermore,carboxyl groups of aspartic- and glutamic acid residues of the Fcfragment can be biotinylated, for example, with Biotin-PEO-Amine,Biotin-PEO-LC-Amine or 5-(biotinamido)-pentylamine.

In another embodiment, other coupling methods may be applied, includinga synthetic method, for example the “click” chemistry (Lutz and Boerner,2008; Moses and Moorhouse, 2007). Carbohydrate groups of the Fc fragmentmay also be biotinylated, in another embodiment, for example, withbiotin hydrazide (Biotin-LC Hydrazide, Pierce catalog No. 21340.

In a preferred embodiment, one or more biotin moieties may be conjugatedto a target protein (e.g., an antibody or an Fc fusion polypeptide). Inone embodiment, a human IgG antibody has an average of 2.4 biotinmoieties/IgG molecule to achieve an increase in plasma half-life. Inother embodiments, a human IgG antibody has an average of 7.2 biotinmoieties/IgG molecule. For human IgG molecules the addition of at least2 or more (e.g., between 2-10) biotin molecules per IgG increases thehalf-life correlating to the number of biotin moieties.

In a preferred embodiment, one or more biotin moieties may be conjugatedto the Fc fragment, to the CH2-CH3-hinge domain of the Fc fragment, orto the hinge region at the CH2-CH3 domain interface of the Fc fragment.The naturally occurring Fc domain is a dimer of the hinge region and thesecond and third constant regions (CH2 and CH3 domain). Biotinylation ofeach monomer at a single site can be sufficient to increase thehalf-life of the antibody or Fc fusion polypeptide, such that at leastone biotin moiety per Fc monomer is sufficient to increase half-life.Thus, in one embodiment, an Fc domain monomer, i.e., a CH2-CH3-hingeregion monomer, comprising a single conjugated biotin moiety can haveincreased half-life, relative to an Fc domain monomer not comprising thebiotin moiety.

In another embodiment, one or more biotin moieties may be conjugated tothe following lysine residues of the human IGHG1 Fc fragment K246, K274,K288, K317, K320, K326, K334, K340, K360, K370, K392, K409, K414 andK439 (UniProtKB/Swiss-Prot P01857; SEQ ID NO: 17) or correspondinglysine residues for other Fc fragments. In a preferred embodiment, thepreferred lysine residues are selected from the group consisting ofK288, K290, K317, K326, K340, K360, and K439.

In another preferred embodiment, the lysine residues K288 and/or K326 ofthe human IGHG1 Fc fragment or the corresponding lysine residues forother Fc fragments are biotinylated.

In another embodiment, the lysine residue to undergo conjugation to abiotin moiety in an Fc fragment is identified using alignment tools andsubsequent alignment of the sequence to the consensus peptide of SEQ IDNO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3

In another embodiment, the corresponding lysine residues are identifiedusing techniques known in the art, for example sequence alignment and 3Dprotein modeling tools.

In other embodiments of the present invention, a mixture of human targetproteins (e.g., an antibody or an Fc fusion polypeptide) isbiotinylated. In one embodiment, a human antibody IgG subtype isbiotinylated. In certain embodiments, a spacer is used for coupling ofbiotin to the target protein. The term “spacer” is used here forchemical group or a short peptide that is between the target protein andthe biotin molecule. Such spacers are also referred to herein as across-linker or linker. In the case of a peptide spacer, this peptidemay have a length from 1 to 20 amino acids comprising a higher number ofalanine and glycines. The spacer may also contain a specific number ofglycines and/or prolines to give flexibility and/or rigidity. Theoptimal spacer may be determined using computational and protein designtools to select favorable spacers.

In certain embodiments of the invention, a spacer is a chemicalmolecule, such as N-hydroxysuccinimide (NHS), NHS-LC, NHS-LC-LC,N-Hydroxysulfosuccinimide (Sulfo-NHS) esters,N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), Succinimidyl6-(3-[2-pyridyldithio]-propionamido)hexanoate (LC-SPDP),Biotin-PEO-Amine, Biotin-PEO-LC-Amine or 5-(biotinamido)-pentylamine. Awide variety of coupling reagents are commercially available (e.g.Pierce). In certain embodiments, the spacer may beSuccinimidyl-6-(biotinamido)hexanoate (NHS-LC biotin), with a size of22.4 Å, or N-Hydroxysuccinimidobiotin (NETS-biotin), with a size of 13.4Å.

In other embodiments, a biotinylated antibody or Fc fusion polypeptidemay optionally comprise one or more additional modifications for furtherincreased persistence in vivo. Examples of such additional modificationsinclude, but are not limited to, PEGylation, glycosylation, sialylation,albumin conjugation, and mutagenesis within the target protein, andmutagenesis of the Fc domain. In the case of amino acid mutagenesis inthe Fc domain, it is known that certain modifications can increasebinding to the FcRn receptor and extend the half life. Examples for suchsubstitutions are N434A and T307/E380/N434 (Petkova et al., 2006c) andothers described in (Dall'Acqua et al., 2006; Hinton et al., 2007;Hinton et al., 2006).

SEQ ID NO: 17: human IGHG1 (UniProtKB/Swiss-Prot P01857)ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGKSEQ ID NO: 1: FNWYVDGVEVHNA K T K PR SEQ ID NO: 2: F K WYVDGVEVHNA K T KPR SEQ ID NO: 3: VSN K ALPAPIE KTherapeutic Applications

In certain embodiments, the present invention provides methods andcompositions for treating various disorders. The methods involveadministering to the individual a therapeutically effective amount ofone or more biotinylated antibodies or biotinylated Fc fusion proteinsas described above. These methods are particularly aimed at therapeuticand prophylactic treatments of animals, and more particularly, humans.

Antibody therapeutics have been proven to be effective for a wide numberof diseases (Carter, 2006). The majority of antibodies developed to dateare to treat cancer: Herceptin for the treatment of breast cancer,Erbitux for colorectal cancer and head and neck cancer, Mylotarg foracute myeloid leukemia, Rituxan, Zevalin and Bexxar for Non-Hodgkinlymphoma, Campath for B-cell chronic lymphocytic leukemia and Avastinfor metastatic colorectal cancer, Non-Small Cell lung cancer andmetastatic breast cancer. Another group of antibodies is used to preventacute kidney transplant rejection (for example Simulect and Zenapax).Others diseases being treated with antibodies are inflammatory diseases,e.g. Remicade and Humira being approved for the treatment of Crohn'sdisease, psoriatic arthritis, rheumatoid arthritis and ankylosingspondylitis and Raptiva for the treatment of psoriasis.

Other indications are asthma (Xolair), multiple sclerosis (Tysabri),cardiovascular (ReoPro for the prevention of clots), infectious diseases(Synagis as prophylactic for prevention of RSV infection in children)and ophthalmology, with Lucentis being approved to treat age-relatedmacular degeneration (AMD). Antibodies are currently being evaluated forthe treatment of amyotrophic lateral sclerosis, polyneuropathy,neurodegenerative diseases, infectious diseases and other disorders.

As described herein, cancer, neurodegenerative diseases,immunodeficiencies, cardiovascular diseases, inflammatory diseases,chronic diseases, eye disorders, transplantation, prenatal disorders,obesity, metabolic disorders, neurodegenerative diseases, infectiousdiseases and autoimmune diseases suitable for treatment by the subjectbiotinylated antibodies or Fc fusion polypeptides include, but are notlimited to, breast cancer, head and neck cancer, colorectal cancer,colon cancer, lung cancer, prostate cancer, brain tumors, glioma,neuroblastoma, ovarian cancer, cervical cancer, melanoma, sarcoma,neoplasia, hepatocellular cancer, pancreatic cancer, renal cell cancer,bladder cancer, thyroid cancer, lymphoma, Non-Hodgkin Lymphoma,leukemia, hematopoietic tumors, oral cancer, esophageal cancer,inflammatory bowel disease, Crohn's disease, psoriatic arthritis,osteoarthritis, asthma, ankylosing spondylitis, psoriasis, age-relatedmacular degeneration, retinopathy, uveitis, hepatitis, transplantrejection systemic lupus erythematosus, insulin resistant diabetes,lysosomal storage disease, myasthenia gravis, polyarteritis, autoimmunethrombocytopenic purpura, cutaneous vasculitis, bullous pemphigoid,pemphigus vulgaris, pemphigus foliaceus, Goodpasture's syndrome,rheumatoid arthritis, Kawasaki's disease, Sjogren's syndrome,osteoporosis, atherosclerosis, coronary heart disease, musculardystrophy, amyotrophic lateral sclerosis, multiple sclerosis, stroke,Alzheimer's and Parkinson disease.

In certain embodiments of such methods, one or more biotinylatedantibodies or biotinylated Fc fusion polypeptides can be administered,together (simultaneously) or at different times (sequentially). In otherembodiments, the biotinylated antibodies or biotinylated Fc fusionpolypeptides of the invention can be administered alone. Alternatively,they may be used in combination with an immunostimulatory agent, animmunomodulator, or a combination thereof. A wide array of conventionalcompounds has been shown to have immunomodulating activities, includingbut not limited to, alpha-interferon, gamma-interferon, tumor necrosisfactor-alpha, or a combination thereof. The present invention recognizesthe effectiveness of conventional therapies for autoimmune diseases,which can be enhanced through the use of one or more biotinylatedantibodies or biotinylated Fc fusion polypeptides of the invention.Alternatively, they may be used in combination with chemotherapy orradiation therapy. Alternatively, they may be used together withchemotherapeutic such as alkylating agents (e.g. cisplatin, carboplatin,oxaliplatin, mechloethamine, cyclophosphamide and chlorambucil),antimetabolites, anthracyclines, plant alkaloids (such as etoposide,teniposide, Paclitaxel and Docetaxel), topoisomerase inhibitors (such asirinotecan, topotecan, amsacrine, etoposide, etoposide phosphate, andteniposide), tyrosine kinase inhibitors (such as imatinib mesylate,dasatinib, lapatinib ditosylate, sorafenib, lestaurtinib, sunitinibmaleate) and the like.

In certain embodiments of the invention, a preparation of biotinylatedpolyclonal antibodies can be administered as polyclonal therapies, whichare effective for use in treatment of infectious diseases, cancer,inflammatory diseases, neurological disorders, autoimmune diseases, andimmunodeficiencies. One such polyclonal therapy is known as IVIg(intravenous administration of heterologous immune globulin), which isalso referred to as IgIV. IVIg is an invaluable FDA-approved palliativetherapy for primary immune deficiencies, such as hypogammaglobulinemia,allogeneic bone marrow transplantation, chronic lymphocytic leukemia,pediatric HIV, idiopathic thrombocytopenic purpura (ITP), Kawasakis'sdisease and kidney transplants. Off label uses of IVIg includetreatments for a wide array of syndromes, including multiple sclerosis,Guillain-Barré syndrome, lupus erythematosus, myasthenia gravis,pemphigus, spontaneous abortion, and severe sepsis(http://en.wikipedia.org/wiki/Intravenous_immunoglobulin). There isevidence that IVIg therapy is beneficial for certain cancers, e.g.prostate cancer, melanoma, colon cancer. IVIg can be combined with othertherapies, for example the combination of IVIg with Rituximab improvesthe chances successful kidney transplantations (Vo et al 2008, N Engl JMed. 2008 Jul. 17; 359(3):242-51).

In further embodiments, the biotinylated antibodies or biotinylated Fcfusion polypeptides of the present invention can be combined with aknown therapy for autoimmune diseases. Examples of such known therapiesfor autoimmune diseases include, but are not limited to, therapies withnonsteroidal anti-inflammatory drugs (NSAID) or corticosteroids. Otherexamples of therapies for autoimmune diseases include periodicadministration of patients with high doses of antibodies.

In certain embodiments of the present invention, a preparation ofbiotinylated antibodies, either monoclonal or polyclonal, can beadministered for passive immunization. Examples for the use of thepreparation of biotinylated antibodies in passive immunization include,but are not limited to, exposure to or infection with viruses, such asHepatitis A virus, Hepatitis B virus, Variola virus, Rabies virus, Ebolavirus, herpes simplex virus, varicella zoster virus, Epstein-Barr virus,tick-borne encephalitis virus, or a cytomegalovirus; exposure to atoxin, such as botulism toxin or snake venom toxin; exposure to orinfection with a bacterial pathogen, such as Bacillus anthracis; and inimmunosuppressive diseases.

Accordingly, the present invention provides methods for the delivery ofa therapeutic antibody or fusion polypeptide across an epithelial layer,the method comprising administering to a subject in need thereof, anantibody or fusion polypeptide comprising an Fc domain, wherein the Fcdomain comprises conjugated biotin, and wherein the biotinylatedantibody or fusion polypeptide is delivered across the epithelialbarrier with increased effectiveness relative to an antibody or fusionpolypeptide lacking biotinylation of the Fc domain

In certain specific embodiments, the biotinylated antibodies and Fcfusion polypeptides of the present invention more efficiently traversemucosal epithelial layers, such as the respiratory and intestinalepithelial layers (Dickinson et al., 1999; Kim et al., 2004; Yoshida etal., 2004). In another embodiment, the biotinylated antibodies and Fcfusion polypeptides more efficiently traverse the corneal or olfactoryepithelial layers. In certain embodiments, the present inventionprovides methods of treating disorders by administering biotinylated IgGantibodies or Fc fusion polypeptides that increase the acid pH-dependentbut not the neutral pH-dependent binding avidity to human FcRn in vitro.

In another specific embodiment, the epithelial layer is an endotheliallayer. In a more specific embodiment, the endothelial layer is a bloodvessel or lymphatic vessel endothelial layer. In such embodiments, theinvention provides methods for enhancing the FcRn-mediated transportacross endothelial layers, such as, in a non-limiting example, from thebloodstream to extravascular sites, thus increasing theirbioavailability in extravascular tissues, for use in the treatment ofsolid tumors.

As used herein, the term “epithelial cells” refers to cells that formthe layer of cells lining the cavities and surfaces of structuresthroughout the body, forming the “epithelium” or “epithelial layer”.Hence, the “epithelial layer”, as referred to herein, comprises thecells that line both the outside (skin) and the inside cavities andlumen of bodies. The epithelial layer can further be sub-divided,according to shape and stratification of the epithelial cells, intosquamous, columnar, cuboidal, stratified, simple, and pseudostratifiedepithelial layers. The epithelial layer is often defined by theexpression of the adhesion molecule e-cadherin, as opposed ton-cadherin, which is used by cells of the connective tissue. Epitheliallayers form the insides of the respiratory system, the gastrointestinaltract, the reproductive and urinary tracts, and constitute the exocrineand endocrine glands. In a non-limiting example, the outer surface ofthe cornea is covered with fast-growing, easily-regenerated epithelialcells.

An “endothelial layer”, as the term is used herein, refers to aspecialized form of epithelium comprising the inner lining of bloodvessels, the heart, and lymphatic vessels, composed primarily of simple,squamous epithelial cells.

Accordingly, in one embodiment, a method is provided for the delivery ofa therapeutic antibody or fusion polypeptide across a fetomaternalorgan, the method comprising administering to a subject in need thereof,an antibody or fusion polypeptide comprising an Fc domain, wherein theFc domain comprises conjugated biotin, and wherein the biotinylatedantibody or fusion polypeptide is delivered across the maternal-fetalinterface with increased effectiveness relative to an antibody or fusionpolypeptide lacking biotinylation of the Fc domain. In a specificembodiment, the fetomaternal organ is the placenta. In such embodiments,the biotinylated antibodies or biotinylated Fc fusion polypeptides ofthe invention can be used for prenatal therapy. Non-limiting examplesfor uses of the biotinylated antibodies or Fc fusion polypeptides inprenatal therapy include lysosomal storage diseases, metabolic diseases,and infectious diseases (Grubb et al., 2008).

The “placenta”, as the term is used herein, is an ephemeral organpresent in placental vertebrates, such as eutherial mammals and sharksduring gestation. The placenta develops from the same sperm and eggcells that form the fetus, and functions as a feto-maternal organ withtwo components, the fetal part (Chorion frondosum), and the maternalpart (Decidua basalis).

Depending on the nature of the combinatorial therapy, administration ofthe biotinylated antibodies or biotinylated Fc fusion polypeptides ofthe invention may be continued while the other therapy is beingadministered and/or thereafter. Administration may be made in a singledose, or in multiple doses. In some instances, administration iscommenced at least several days prior to the conventional therapy, whilein other instances, administration is begun either immediately before orat the time of the administration of the conventional therapy. Incertain specific embodiments, the therapeutic biotinylated agent isadministered by subcutaneous, intravenous, intranasal, parenteral,transdermal, intratracheal, intravenous, intramuscular, intracranial,intrathecal or intravitreal injection; by oral administration, eyedrops, pessary or inhalation.

Pharmaceutical Compositions and Modes of Administration

An object of the present invention, in part, is to provide improvedtherapeutic regimens, wherein administration of biotinylated antibodiesand Fc fusion polypeptides, allows for less frequent dosageadministrations and reduced effective doses, relative to antibodies andFc fusion polypeptides not comprising a biotin moiety. In certainembodiments, the subject biotinylated antibodies or biotinylated Fcfusion polypeptides of the present invention are formulated with apharmaceutically acceptable carrier. The biotinylated antibodies orbiotinylated Fc fusion polypeptides can be administered alone or as acomponent of a pharmaceutical formulation (composition). They may beformulated for administration in any convenient way for use in human orveterinary medicine. Wetting agents, emulsifiers and lubricants, such assodium lauryl sulfate and magnesium stearate, as well as coloringagents, release agents, coating agents, sweetening, flavoring andperfuming agents, preservatives and antioxidants can also be present inthe compositions.

The subject formulations of the present invention include those suitablefor oral, dietary, topical, parenteral (e.g., intravenous,intraarterial, intramuscular, subcutaneous injection), inhalation (e.g.,intrabronchial, intranasal, or oral inhalation; intranasal drops),rectal, and/or intravaginal administration. Other suitable methods ofadministration can also include rechargeable or biodegradable devicesand slow release polymeric devices. The pharmaceutical compositions ofthis invention can also be administered as part of a combinatorialtherapy with other agents (either in the same formulation or in aseparate formulation).

The formulations may conveniently be presented in unit dosage form andmay be prepared by any methods well known in the art of pharmacy. Theamount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will vary depending upon thehost being treated and the particular mode of administration. The amountof active ingredient which can be combined with a carrier material toproduce a single dosage form will generally be that amount of thecompound which produces a therapeutic effect.

In certain embodiments, methods of preparing these formulations orcompositions include combining another type of immune-modulating agentand a carrier and, optionally, one or more accessory ingredients. Ingeneral, the formulations can be prepared with a liquid carrier, or asolid carrier, or both, and then, if necessary, shaping the product.

Formulations for oral administration may be in the form of capsules,cachets, pills, tablets, lozenges (using a flavored basis, usuallysucrose and acacia or tragacanth), powders, granules, or as a solutionor a suspension in an aqueous or non-aqueous liquid, or as anoil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup,or as pastilles (using an inert base, such as gelatin and glycerin, orsucrose and acacia), and/or as mouth washes and the like, eachcontaining a predetermined amount of one or more subject antibodies asan active ingredient.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups,and elixirs. In addition to the active ingredient, the liquid dosageforms may contain inert diluents commonly used in the art, such as wateror other solvents, solubilizing agents and emulsifiers, such as ethylalcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils(in particular, cottonseed, groundnut, corn, germ, olive, castor, andsesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycolsand fatty acid esters of sorbitan, and mixtures thereof. Besides inertdiluents, the oral compositions can also include adjuvants such aswetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming, and preservative agents. Suspensions, inaddition to the active compounds, may contain suspending agents such asethoxylated isostearyl alcohols, polyoxyethylene sorbitol, and sorbitanesters, microcrystalline cellulose, aluminum metahydroxide, bentonite,agar-agar and tragacanth, and mixtures thereof.

Methods of the invention can be administered topically, for example, tothe skin. The topical formulations may further include one or more ofthe wide variety of agents known to be effective as skin or stratumcorneum penetration enhancers. Examples of these include 2-pyrrolidone,N-methyl-2-pyrrolidone, dimethylacetamide, dimethylformamide, propyleneglycol, methyl or isopropyl alcohol, dimethyl sulfoxide, and azone.

Additional agents may further be included to make the formulationcosmetically acceptable. Examples of these are fats, waxes, oils, dyes,fragrances, preservatives, stabilizers, and surface active agents.Keratolytic agents such as those known in the art may also be included.Examples are salicylic acid and sulfur.

Dosage forms for the topical or transdermal administration includepowders, sprays, ointments, pastes, creams, lotions, gels, solutions,patches, and inhalants. The subject antibodies may be mixed understerile conditions with a pharmaceutically acceptable carrier, and withany preservatives, buffers, or propellants which may be required. Theointments, pastes, creams and gels may contain, in addition to abiotinylated antibody or Fc fusion polypeptide, excipients, such asanimal and vegetable fats, oils, waxes, paraffins, starch, tragacanth,cellulose derivatives, polyethylene glycols, silicones, bentonites,silicic acid, talc and zinc oxide, or mixtures thereof.

Pharmaceutical compositions suitable for parenteral administration maycomprise one or more biotinylated antibodies or biotinylated Fc fusionproteins in combination with one or more pharmaceutically acceptablesterile isotonic aqueous or nonaqueous solutions, dispersions,suspensions or emulsions, or sterile powders which may be reconstitutedinto sterile injectable solutions or dispersions just prior to use,which may contain antioxidants, buffers, bacteriostats, solutes whichrender the formulation isotonic with the blood of the intended recipientor suspending or thickening agents. Examples of suitable aqueous andnonaqueous carriers that may be employed in the pharmaceuticalcompositions of the invention include water, ethanol, polyols (such asglycerol, propylene glycol, polyethylene glycol, and the like), andsuitable mixtures thereof, vegetable oils, such as olive oil, andinjectable organic esters, such as ethyl oleate. Proper fluidity can bemaintained, for example, by the use of coating materials, such aslecithin, by the maintenance of the required particle size in the caseof dispersions, and by the use of surfactants. These compositions mayalso contain adjuvants, such as preservatives, wetting agents,emulsifying agents and dispersing agents. Prevention of the action ofmicroorganisms may be ensured by the inclusion of various antibacterialand antifungal agents, for example, paraben, chlorobutanol, phenolsorbic acid, and the like. It may also be desirable to include isotonicagents, such as sugars, sodium chloride, and the like into thecompositions.

In addition, prolonged absorption of the injectable pharmaceutical formmay be brought about by the inclusion of agents which delay absorption,such as aluminum monostearate and gelatin. Injectable depot forms aremade by forming microencapsule matrices of one or more antibodies inbiodegradable polymers such as polylactide-polyglycolide. Depending onthe ratio of drug to polymer, and the nature of the particular polymeremployed, the rate of drug release can be controlled. Examples of otherbiodegradable polymers include poly(orthoesters) and poly(anhydrides),collagen, hydrogel, chitosan and alginate. Depot injectable formulationsare also prepared by entrapping the drug in liposomes, polymericnanoparticles or microemulsions which are compatible with body tissue.

In certain embodiments, the pharmaceutical composition is administeredby subcutaneous, intravenous, intranasal, parenteral, transdermal,intracheal, intravenous, intramuscular, intracranial, intrathecal orintravitreal injection; by oral administration, eye drops, pessary;inhalation or implantation.

EXEMPLIFICATION

The invention now being generally described will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

Example 1: Biotinylation of Human Immunoglobulin G

Purified human immunoglobulin G (hIgG) (Gammagard, Baxter) wasconjugated with biotin by first diluting the hIgG stock in 50 mM sodiumphosphate buffer, pH 7.2 with the same buffer to 2.2 mg/ml, or 1.37×10⁻⁵M. N-hydroxysuccinimidobiotin (EZ-Link™ NHS-biotin, Pierce cat. #20217)was allowed to come to room temperature, then a 3.42×10⁻³M solution ofNHS-biotin in DMSO (10× strength) was produced. One volume of 3.42×10⁻³M(10×) NHS-biotin was added to 9 volumes of 1.37×10⁻⁵M hIgG to achieve a25-fold excess molar concentration of NHS-biotin. The reaction wasperformed for 30 minutes at room temperature, then 1 volume of 1 Mglycine, pH 7.2 was added to stop the reaction. The reactants were twicedialyzed in 5 liters of 50 mM sodium phosphate buffer, pH 7.2 at 4° C.The hIgG-biotin solution was sterile filtered using a 0.45 μm poresyringe filter and stored at 4° C. To determine the biotin:hIgG ratiothe EZ Biotin Quantitation Kit (Pierce cat. #28005) in the microplateformat method was used, assaying all samples in triplicate. 100 μl ofpure water was added to each No-Weigh™ HABA/Avidin Premix. 160 μl of 50mM sodium phosphate buffer, pH 7.2 was added into wells of a microplate.20 μl of the HABA/Avidin Premix solution was added to the PBS in thewells. The average A_(500 nm) HABA/avidin equaled 0.542. 20 μl ofhIgG-biotin, or biotinylated standard included in the kit was added. Theaverage A_(500 nm) HABA/avidin/biotin equaled 0.501. The averageA_(280 nm) of hIgG-biotin was determined to be 2.044 and divided by theextinction coefficient 1.4 to determine the protein concentration as1.46 mg/ml. According to the Beer Lambert Law: A_(λ)=ε_(λ)bC, where A isthe absorbance of a sample at a particular wavelength (λ). ε is theextinction coefficient at the wavelength λ. For HABA/avidin at 500 nm,pH 7.0, the extinction coefficient equals 34,000 ml/(M−¹cm⁻¹). b is thelight's path length through the sample expressed in cm. C is theconcentration of the sample expressed in mmol/ml. Applied this equationto the values obtained above,

$\frac{{mmol}\mspace{14mu}{biotin}}{{mmol}\mspace{14mu}{hIgG}} = {\frac{\left( {\Delta\; A_{500\mspace{14mu}{nm}}\text{/}\left( {34\text{,}000\mspace{14mu}{ml}\text{/}\left( {M^{- 1}{cm}^{- 1}} \right) \times b} \right)} \right)(20)}{\left( {A_{280\mspace{14mu}{nm}}\text{/}1.4} \right)\text{/}146\text{,}000\mspace{14mu} g\text{/}{mol}} = {\frac{\left( {0.041\text{/}\left( {34\text{,}000 \times 0.5} \right)} \right) \times 20}{\left( {2.044\text{/}1.4} \right)\text{/}146\text{,}000} = 4.8}}$the biotin:hIgG ratio, where ΔA_(500 nm) nm equals A₅₀₀ HABA/avidinminus A₅₀₀ HABA/avidin/biotin, 20 was a correction factor for thecombination 2-fold predilution and 10-fold dilution of the hIgG-biotinin the hIgG-biotin/HABA/avidin mixture, and 146,000 g/mol is themolecular weight of hIgG.

Example 2: Determination of hIgG-Biotin Half Life In Vivo

To determine the effect of biotinylation on the in vivo half life ofhIgG, C57BL/6 mFcRn−/−hFcRn+/Tg Line 276 mice (3-5 mice/group) weregiven intraperitoneal injections of 100 μg of hIgG with, or without,biotin conjugation at day −3. C57BL/6 mFcRn−/−hFcRn+/Tg Line 276 mice dolack the mouse FcRn gene and do express the human FcRn. For a moredetailed description of the mouse model, see (Petkova et al., 2006a).Plasma was obtained 3 days after tracer injection (to avoid the alphaphase of hIgG degradation), at day 0, 7, 14, 21, and 28, by collectingeither 25 μl or 75 μl of blood from the retro-orbital sinus intocapillary tubes and transferring to 1.5 ml microcentrifuge tubescontaining 2 μl of PBS with 10,000 U/ml heparin. Tubes were centrifugedat 10,000 rpm for 5 minutes at 4° C. in an Eppendorf 5417R centrifuge(New York). Plasma samples were transferred to 96 well round bottompolypropylene storage plates (Corning cat. #3365), plug seal coveredwith Microplate Storage MattII covers (Corning cat. #3092) and stored at−70° C. The storage plate was sandwiched between freezer packs tominimize volume loss to by evaporation.

Quantitation of hIgG by ELISA

hIgG specific ELISA of plasma samples was performed using 96 well highbinding ELISA plates (Greiner cat #655061) coated with mouse anti-humanIgG-Fc (Southern Biotech cat. #9040-01) diluted to 0.5 μg/100 μl/well inDulbecco's phosphate buffered saline, pH 7.2 (DPBS, Hyclone cat.#SH30013) overnight at 4° C. Plates were hand washed using a manifoldwith 96 nozzles connected to a 30 ml syringe (Bel Art Vaccupette/96) 2times 300 μl per well using DPBS with 0.05% Tween 20 and 0.05% sodiumazide (ELISA Wash). Wash was removed by flicking plates into a sink andblotting the plates on paper towels. Plates were blocked with 300μl/well of ELISA Block (ELISA Wash with 1% bovine serum albumin (BSA,Fitzgerald cat. #30-AB75)) for a minimum of 1 hour at room temperature.Plasma samples were diluted 1/200 with diluent (same as ELISA Block).HIgG and hIgG-biotin were diluted 2-fold from 1000 ng/ml down to 1 ng/mlto act as standards. Block was removed by flicking and blotting asbefore. 100 μl of diluted standards and plasma as well as diluent only(to act as blanks) were transferred into the coated and blocked ELISAplates. Plates were incubated at 37° C. for 1 hour, then washed asbefore 3 times 300 μl/well. Added 100 μl/well of mouse anti-humankappa-AP (Southern Biotech cat. #9220-04) diluted 1/1000 in diluent andincubated at 37° C. for 1 hour, then washed as before 3 times 300μl/well. Added 100 μl/well of 1 mg/ml p-nitrophenyl phosphate (SigmaCat. #N2765) in Substrate Buffer (20 mM sodium bicarbonate, 24 mM sodiumcarbonate, 7 mM magnesium chloride hexahydrate, pH 8.6). Read A_(405 nm)at 30 minutes, or when the maximum optical density exceeded 1. Data isplotted as the percent tracer remaining compared to the day 0 plasmatracer concentration. Half-life was calculated using the formula:

$t_{1\text{/}2} = {\frac{{Log}\mspace{14mu} 0.5}{{Log}\mspace{14mu} A_{e}\text{/}A_{0}} \times t}$where t_(1/2) is the half life of the tracer, A_(e) is the amount oftracer left, A₀ is the original amount of tracer at day 0, and t is theelapsed time.

This data indicates that when biotin is conjugated to the availablelysine residues on hIgG and injected into the peritoneal cavity ofC57BL/6 mFcRn−/−hFcRn+/Tg Line 276 mice, hIgG-biotin has a longerhalf-life compared to hIgG.

Analysis by a Second ELISA Assay

In addition to the mouse anti-human kappa-AP detection reagent describedabove, Streptavidin-horse radish peroxidase (SAv-HRP, Southern Biotechcat. #7100-05) was used as a second detection reagent. First, a hIgGELISA was performed as described above, except azide-free buffers wereused to preserve the SAv-HRP activity. After the alkaline phosphatase(AP) activity was measured, the plates were washed as described above 1times 300 μl/well. Added 100 μl/well SAv-HRP diluted 1/1000 inazide-free diluent right into the very same washed plates that had justhad their mouse anti-kappa-AP activity measured, and incubated 1 hour at37° C. Plates were washed as described above 3 times 300 μl/well. Added100 μl/well 3,3′,5,5′ tetramethylbenzidine (TMB, Pierce cat #34028) as ahorseradish peroxidase (HRP) substrate. After 30 minutes of colordevelopment, added 50 μl/well of 2 M sulfuric acid to stop the reaction,and measured the A_(450 nm).

Using either method for the ELISA, a) detection of biotinylated hIgG viaits kappa chain (using mouse anti-human kappa-AP), or b) via the biotingroup (SAv-HRP), the curves obtained for hIgG-biotin are identical andshow increased protection observed for hIgG-biotin when compared tohIgG. This demonstrates that biotinylation of hIgG increases its plasmahalf-life (see FIG. 1 and FIG. 2).

Example 3: Correlation of Degree of Biotinylation to Plasma Half-Life

To determine what degree of biotin conjugation was necessary to effectan increased half life, hIgG was labeled as described in Example 1, butwith the reaction performed on ice to slow the rate to allow moreprecise labeling ratios, and the length of reaction was varied to obtainthe biotin:hIgG ratios 1.2, 2.4, 4.6, and 7.2. As a model to analyze theplasma half life in vivo, we have used a mouse model expressing thehuman FcRn while lacking the mouse FcRn receptor on the C57BL/6background (C57BL/6 FcRn−/−; hFcRn Tg276), which is described in(Petkova et al., 2006a). 100 μg of each of these hIgG-biotin tracers andhIgG were intraperitoneally injected into C57BL/6 mFcRn−/−hFcRn+/Tg Line276 mice (5 mice/group) at day −3. Plasma was collected from mice on day0, 7, 14, 21, and 28, and measured by hIgG ELISA as described in Example2.

The conjugation of biotin to available lysine residues on hIgG increasedthis tracer's half-life in a dose dependent manner. When spread over allavailable lysine residues on hIgG, 2.4 biotin molecules per hIgG was theminimum number necessary to observe a significant increase in half-lifecompared to hIgG (p=0.012) as shown in FIG. 3.

Example 4: Comparison of Biotinylation of Fc Fragment to hIgG

hIgG Fc (Bethyl Laboratories cat. #P80-204) was biotin conjugated asdescribed in Example 1 at room temperature for 30 minutes. Thebiotin:hIgG Fc ratio was quantified to be 6.2 using the method describedin Example 3 assuming that the hIgG Fc fragment has a molecular weightequal to 50,000 g/mol. 100 μg of hIgG Fc-biotin, or hIgG Fc tracers wereintraperitoneally injected into C57/BL6 mFcRn−/−hFcRn+/Tg Line 276 mice(5 mice/group) at day −3. Plasma was collected from mice and measured byhIgG ELISA as described in Example 2. hIgG Fc-biotin shows a longerplasma half-life than the hIgG Fc alone, indicating that lysines locatedon the Fc region of hIgG add to the protection of hIgG when biotinylated(see FIG. 4).

Example 5: hIgG-Biotin Half-Life Determination in the Absence of FcRn

Another approach to determining the mechanism for the increasedhalf-life of hIgG-biotin compared to hIgG would be to manipulate theother side of the hIgG-FcRn equation. To accomplish this, hIgG-biotinand hIgG were used as tracers in C57BL/6 mFcRn−/−mice which lack boththe mouse, and human FcRn (Roopenian et al., 2003). hIgG-biotin and hIgGFc tracers were intraperitoneally injected into C57BL/6 mFcRn−/−mice (5mice/group) at day −1. Plasma was collected from mice on day 0, 1, 2, 4,and 6 and measured by hIgG ELISA as described in Example 2.

In the absence of either human or mouse FcRn to protect the hIgGtracers, the addition of biotin to hIgG did not augment the half-life ofthe hIgG-biotin. This suggests that biotin conjugated to hIgG, a ligandof FcRn, interacts with the hFcRn in such a way to increase itsprotection from the lysosomal degradation pathway.

Example 6: Comparison of Biotinylated Serum Albumin to Serum Albumin

Serum albumin is the only other ligand known to bind FcRn other thanIgG. Therefore, it was determined if biotinylation of human serumalbumin (HSA) extends its half-life. HSA (Sigma, >96% purity) was biotinconjugated as described in Example 1 at room temperature for 30 minutes.The biotin:HSA ratio was quantified to be 6.6 using the method describedin Example 1. To determine the half-life, 1 mg of HSA-biotin and HSAtracers were intraperitoneally injected into C57BL/6 mFcRn−/−hFcRn+/TgLine 276 mice (5 mice/group) at day −1. Plasma was collected from miceon day 0, 1, 2, 4, 6, and 8 and measured by HSA ELISA.

An HSA ELISA was performed by coating ELISA plates with rabbitanti-human albumin (US Biological cat. #A1327-46) that was diluted to0.5 μg/100 μl in 50 mM sodium bicarbonate buffer, pH 9.6 and incubatedovernight at 4° C. Plates were hand washed 2 times 300 μl per well usingDPBS with 0.05% Tween 20 and 0.05% sodium azide (ELISA Wash). Wash wasremoved by flicking plates into a sink and blotting the plates on papertowels. Plates were blocked with 300 μl/well of ELISA Block (ELISA Washwith 1% bovine serum albumin (BSA, Sigma cat. #A7284)) for a minimum of1 hour at room temperature. Plasma samples were diluted 1/200 and 1/800with diluent. HSA and HSA-biotin were diluted 2-fold from 1000 ng/mldown to 1 ng/ml to act as standards. Block was removed by flicking andblotting as before. 100 μl of diluted standards and plasma as well asdiluent only (to act as blanks) were transferred into the coated andblocked ELISA plates. Plates were incubated at 37° C. for 1 hour, thenwashed as before 3 times 300 μl/well. Added 100 μl/well of goatanti-human HSA-AP (Bethyl Laboratories cat. #A80-229AP) diluted 1/200 indiluent and incubated at 37° C. for 1 hour, then washed as before 3times 300 μl/well. Added 100 μl/well of 1 mg/ml p-nitrophenyl phosphate(Sigma Cat. #N2765) in Substrate Buffer. Read A_(405 nm) at 30 minutes,or when the maximum optical density exceeded 1. Data is plotted as thepercent tracer remaining compared to the day 0 plasma tracerconcentration. Biotinylation does not alter the half-life of HSA invivo.

Example 7: Biotin and not the Spacer Increases the Half-Life of IgG

Biotinylation was conventionally applied usingN-hydroxysuccinimidobiotin (NHS-biotin) chemistry in which the NHS-esterreacts with primary amine and the epsilon amines of lysine. Toinvestigate the importance of the biotin group, the half-life ofNHS-biotin derivatized hIgG was compared to that of hIgG which wasderivatized with C6-succinimidyl 4-formylbenzoate (C6-SFB). Biotin-NHS(Pierce cat. #20217) and C6-SFB (Pierce cat. #22423) have the samelength of 13.5 Å. The NHS chemistry used for both reagents allows aconjugation to epsilon amines of lysine. C6-SFB does not contain biotin,but instead the ring structure 4-formylbenzoate (see FIG. 7). LC-biotinand C6-SFB were conjugated to hIgG in parallel using the same methodused to conjugate biotin to hIgG as described in Example 1. Afterreacting hIgG with C6-SFB, it was dialyzed. A reductive aminationreaction using ethanolamine and sodium cyanobromohydride converted thealdehyde reactive end of the spacer C6-SFB to an unreactive hydroxyl.After adding 10 μl ethanolamine (Sigma cat. # E9508) to half of thehIgG-C6-SFB (1.7 ml) to get 100 mM ethanolamine. In a hood whilevortexing, 17 μl of 5 M sodium cyanoborohydride in 1 M NaOH (Sigma cat.#296945) were added and incubated for 15 minutes at room temperature.The sample was dialyzed, again, and sterile filtered. The biotinconjugate had its biotin:hIgG ratio measured using the same method inExample 1, resulting in a ratio of 7.6. These hIgG conjugated tracersand unconjugated hIgG were intraperitoneally injected into C57BL/6mFcRn−/−hFcRn+/Tg Line 276 mice (5 mice/group) at day −3. Plasma wascollected from mice and measured by hIgG ELISA as described in Example2. FIG. 12 shows that hIgG-C6-SFB, with the same overall length asbiotin, but lacking a biotin moiety, did not alter the half-life whencompared to hIgG, suggesting that the biotin moiety is important for theinduced enhancement of hIgG half-life.

Example 8: Influence of Spacer on the Half-Life of Biotinylated IgG

Two spacers were compared, N-hydroxysuccinimidobiotin (NHS-biotin) andNHS-LC-biotin (Pierce cat. #21336) and see FIG. 7. NHS-biotin is 13.5 Åand for NHS-LC-biotin 22.4 Å long. The conjugation to human IgG (hIgG)was performed as described in Example 1 and the biotin:hIgG ratio forboth linkers was approximately 7.6. These hIgG conjugated tracers andunconjugated hIgG were intraperitoneally injected into C57BL/6mFcRn−/−hFcRn+/Tg Line 276 mice (5 mice/group) at day −3. Plasma wascollected from mice and measured by hIgG ELISA as described in Example2. When calculating the half-life hIgG-biotin yielded 10.5 days versushIgG-LC-biotin 8.8 days versus the unconjugated hIgG control with 7.5days (see table 1 and FIG. 8). This shows that with both spacers animproved half-life can be achieved, with the shorter spacer performingbetter.

TABLE 1 Measurement of half-life in the C57BL/6 mFcRn−/−hFcRn+/Tg Line276 mouse model Half-life IgG type (days) hIgG 7.5 hIgG-LC-biotin 8.8(NHS-LC-biotin) hIgG-biotin 10.5 (NHS-biotin)

Example 9: Determination of hIgG1 Half-Life with Biotin Conjugation

Polyclonal hIgG-biotin and hIgG, prepared as described in Example 1, wasintraperitoneally injected into hemizygous C57BL/6J mFcRn−/−hFcRn+/TgLine 276 mice (5 mice/group) and homozygous C57BL/6J mFcRn−/−hFcRn Tg/TgLine 276 mice (5 mice/group) at day −3. Plasma was collected from miceon day 0, 7, 14, 21, and 28, diluted 1/100, and measured by ELISA forhIgG1 as described in Example 1, except mouse anti-human IgG1 Fc(Southern Biotech cat. #9052-01) was used at 1/200 dilution to capture,and hIgG1 (Calbiochem cat. #400120) was used as a standard.Biotinylation of hIgG1 extends the half-life of the hIgG1 portion ofhIgG from 4.5±0.7 days to 10.0±0.7 days for the hemizygous and from7.0±1.6 days to 17.0±1.3 days for the homozygous group as shown in FIG.9.

Example 10: Determination of the Half-Life of Biotinylated Mouse IgG1 InVivo

Mouse IgG1 (mIgG1) anti-DNP mAb (1B7.11: Kimura et al. Immunology 1986,59, 235-238) was Protein G purified from ascites, and biotin conjugatedas described in Example 1 to yield a biotin:mIgG1 ratio of 4.6. Todetermine the in vivo half-life of mIgG1-biotin and mIgG1 tracers, 100μg of either was intraperitoneally injected into C57BL/6J, or C57BL/6JmFcRn−/−mice (5 mice/group) at day −1. Plasma was collected from mice onday 0, 1, 2, 4, and 38 and measured by ELISA as described in Example 1,except that bovine gamma globulin-DNP (Calbiochem cat. #324111) was usedat 0.5 μg/100 μl/well to capture, goat anti-mouse kappa-AP (SouthernBiotech cat. #1050-04) was used at 1/1000 to detect, and mIgG1 (1B7.11),or mIgG1-biotin were used to standardize. Biotinylation of mIgG1 doesnot extend its half life when used as a tracer in either C57B/6JmFcRn−/−, or C57B/6J wild type mice. FIG. 10 shows that biotinylationdoes not improve the half-life when only murine FcRn receptor is presentor if no FcRn receptor is present suggesting that biotin is involved inthe interaction with the human but not the mouse FcRn receptor.

Example 11: Determination of Biotinylated hIgG1 Chimeric AntibodyHalf-Life In Vivo

We tested if biotinylation can extend the half life of human chimericantibodies using the chimeric antibody HuLys11, as an example (Foote andWinter, 1992). HuLys11 is composed of a human IgG1 Fc part and mouseheavy and light chain complementarity determining region (CDR) 1, 2, and3 residues derived from the mouse mAb D1.3, which give the chimericantibody its hen egg lysozyme (HEL) specificity. HuLys11 wasbiotinylated using the method described in Example 1. In a separatereaction, HuLys11 was also conjugated in parallel with a pH sensitivederivative of biotin called 2-iminobiotin, a cyclic guanidino analog ofbiotin. Iminobiotin binds streptavidin, like biotin, however it bindsbest at pH 9.5. As an example, only 2.5% of an iminobiotinylated protein(fetuin) bound to an avidin affinity column at pH 7.5, while 93% boundat pH 9.5 (Orr, 1981). The biotin:HuLy11 ratio was determined to be 4.5using the method as described in Example 1. Due to iminobiotinpreferentially binding to streptavidin at pH 9.5, or greater, the pH 7.5buffers used to quantitate biotin in Example 1 would not allow accurateiminobiotin quantitation. An ELISA method similar to the one used inExample 2 was implemented to quantitate iminobiotin: HEL (Sigma cat.#L6876) at 0.5 μg/100 μl 50 mM sodium carbonate, pH 9.5 per well of the96 well ELISA plate was used to capture, streptavidin-AP (SouthernBiotech cat. #7100-04) diluted 1/1000 in 50 mM sodium carbonate, pH 9.5,0.05% Tween 20, and 0.05% bovine serum albumin (BSA) was used to detect,and HuLys11-biotin was used as a standard. The ELISA plate was washed asdescribed as described in Example 1, except 50 mM sodium carbonate, pH9.5, with 0.05% Tween 20 was used as the wash buffer.HuLys11-iminobiotin was determined to have the same ratio asHuLys11-biotin: 4.5. HuLys11-biotin, HuLys11-iminobiotin, and HuLys11were intraperitoneally injected (100 μg/mouse) into C57BL/6mFcRn−/−hFcRn+/Tg Line 276 mice (5 mice/group) at day −3. Plasma wascollected from mice on day 0, 7, 14, 21, and 28, and measured using amodified ELISA similar to the method used in Example 2: HEL at 0.5μg/100 μl PBS/well was used to capture, mouse anti-human kappa-AP wasused to detect, and HuLys11 was used as a standard. Apparently due tothe chimeric nature of HuLys11, this tracer had a half-life (23±0.2days) that was significantly lower than the half-life determined forhIgG1 in Example 9 (4.5±0.7 days, p=0.001). Biotinylation significantlyaugmented the half-life of HuLys11 to 4.3±0.6 days (p=0.02). In spite ofthe high pH-dependent nature of iminobiotin binding to streptavidin,HuLys11-iminobiotin also had a half-life (3.4±0.2 days), which wassignificantly longer than for HuLys11 alone (p=0.00003) as shown in FIG.11.

Example 12. Half-Life Determination of Biotinylated Fc Fragment ofHumanized hIgG1 Antibody In Vivo

Herceptin (trastuzumab) is a humanized antibody carrying a hIgG1 Fc. Todetermine the effect of biotinylation on the half-life of this hIgG1 Fcfragment, the Fc fragment of Herceptin was purified, biotinylated,administered in vivo, and assayed by ELISA. 100 mg of Protein G purifiedHerceptin was digested in 20 mM sodium phosphate, pH 7.2, 10 mM EDTA, 20mM cysteine using 5 ml of 50% immobilized papain slurry (Pierce Cat.#20341 with 0.25 mg papain/ml of gel) with Herceptin at 20 mg/ml(enzyme:substrate ratio of 1:160) for 4 hours at 37° C. with tuberotation. This preparation was centrifuged to recover digested Herceptinfragments as supernatant. The papain digested Herceptin was dialyzedagainst 10 mM sodium borate, pH 9.5 extensively. Herceptin fragmentswere diluted with 10 mM sodium borate, pH 9.5 (2 mg/ml protein), thenadded to 30 ml of regenerated DEAE-cellulose (Sigma D0909) that had beenequilibrated with 10 mM sodium borate, pH 9.5. After rotation to mix for1 hour, the DEAE-cellulose was centrifuged and the unbound fraction wasdiscarded as F(ab)2. The DEAE-cellulose was exhaustively washed with 10mM sodium borate, pH 9.5. The bound Herceptin Fc fragment was theneluted by adding 100 mM sodium citrate, pH 4.0. An Amicon Ultra-15(Millipore UFC903024) was used to concentrate the Fc Fragment, which wasthen washed with 25 mM sodium phosphate, pH 7, and sterile filtered inpreparation for injection. The Herceptin Fc fragment (>99% purity) wasassessed by BCA protein assay to determine protein quantity, and bySDS-PAGE.

The Herceptin Fc fragment was biotin conjugated as described in Example1 at room temperature for 30 minutes. The biotin:hIgG1 Fc ratio wasquantified to be 4.4 using the method described in Example 1, assumingthat the Herceptin Fc fragment has a 50,000 molecular weight. 100 μg ofHerceptin Fc-biotin, or unconjugated Fc tracers were intraperitoneallyinjected into C57/BL6 mFcRn−/−hFcRn+/Tg Line 276 mice (5 mice/group) atday −3. Plasma was collected from mice on day 0, 3, 7, 11, 14 andmeasured by hIgG ELISA as described in Example 2 with the exception thatgoat anti-human IgG-alkaline phosphatase (Southern Biotech 2040-04)diluted 1/1000 in diluent was used for detection. The results in FIG. 15show that the unconjugated Herceptin Fc fragment half life (2.4±0.4) andbiotinylated half life (5.6±1.3) were similar to the hIgG Fc fragmenthalf life values and augmentation shown in Example 4.

Example 13: Determination if Biotinylation of hIgG Results Lowers theIC50 when Competing for Human FcRn Binding In Vitro

It is known in the art that alterations in IgG antibodies that increasebinding to FcRn in an acidic but not in a neutral pH environment canextend their half-life in vivo. Therefore, a primary mechanism, withoutwishing to be limited by a theory, which could explain the extension ofthe half-life of biotinylated hIgG is that the biotin modificationenhances its binding avidity for hFcRn at an acidic pH. A flow-cytometrycell-based competitive binding assay, used successfully in the past,relies on binding labeled ligands to hFcRn on the plasma membrane, whichshould at least partially mimic the membrane dynamics of IgG/FcRnengagement in vivo. This cellular assay used the human 293 cell linethat expresses a GFP-hFcRn construct lacking the endosomal targetingdomain, thus diverting GFP-hFcRn expression to the plasma membrane(Petkova et al., 2006a). In a competitive binding assay, 5×105 cells/25μl/test were incubated with a constant concentration of hIgG labeledwith Alexa Fluor 647 (40 μg/ml), in competition with variousconcentrations (two fold serial dilutions from 320 μg/ml to 1.25 μg/ml,or 0 μg/ml) of biotinylated or unconjugated hIgG (competitors lacked afluorescent label) in FACS buffer (PBS containing 1% BSA, 0.05% sodiumazide) at pH 5.8, or 7.2 on ice for 2 hours. The cells were washed onetime with 2 ml FACS Buffer at pH 5.8, or pH 7.2 and resuspended in 500μl FACS buffer containing propidium iodide (0.2 μg/ml) to allow analysisof viable cells. Samples were analyzed for inhibition of binding ofhIgG-Alexa Fluor 647 to hFcRn by competing hIgG using a FACSCalibur™ (BDBiosciences) flow cytometer. The Alexa Fluor 647 mean fluorescentintensity (MFI) of GFP gated events were plotted against competitorconcentrations (FIG. 15). The MFI value at 50% inhibition was calculatedfrom the formula:MFI50% inhibition=(MFImax−MFImin)/2+MFIminwhere the MFImax is the MFI of cells at pH 5.8 with Alexa Fluor 647-hIgGwithout competitor and the MFImin is the MFI of cells at pH 7.2 withAlexa Fluor 647-hIgG at all competitor concentrations. The IC50 valueswere interpolated from the MFI value at 50% inhibition (102.7) to yieldIC50 for hIgG-biotin (5.4×10−6 M) and hIgG (1.14×10−5 M) in FIG. 15. Theresults show that biotinylated hIgG has an approximate two folddecreased IC50 for hFcRn at an acidic pH 5.8, but not at a neutral pH7.2 where no specific binding correlated to GFP intensity.

Example 14: Identification of Specific Biotinylated Lysine Residues inthe Fc Fragment of the Chimeric Monoclonal Antibody HuLys11

The chimeric HuLys11 is comprised of a human IgG1 Fc fragment and amouse heavy and light chain complementarity determining region (CDR) 1,2, and 3 (Foote and Winter, 1992). HuLys11 was biotinylated with the pHsensitive derivative of biotin, 2-iminobiotin, as described in Example11 and functionally validated to confer a half-life extension asillustrated in FIG. 11. Mass spectrometry (MS) analysis was performed toidentify the lysine residues of HuLys11 that are biotinylated by thismethod. 300 micrograms of iminobiotinylated HuLys11 was dialyzed against50 mM ammonium bicarbonate, pH 9.5. This preparation was reduced byadding 12.5 μl 200 mM tributylphosphine in 1-methyl-2-pryrolidone (TBP)from the ProteoPrep Reduction and Alkylation Kit (Sigma, Cat. #PROT-RA). The final concentration of TBP was 5 mM. This preparation wasthen incubated for 60 minutes at room temperature (RT), and thenalkylated by adding 0.5 M iodoacetamide (15 mM final concentration) andincubated for 90 minutes at RT. The excess iodoacetamide was quenchedthe adding 200 mM tributylphosphine in 1-methyl-2-pryrolidone andincubated for an additional 15 minutes at RT. Acetonitrile was thenadded to 4.5% v/v and HC1 acidified trypsin was added to achieve a 1:40trypsin to HuLys11-IB ratio. The samples were then digested byincubation at 37° C. overnight (˜18 hours), and 50 mM ammoniumbicarbonate, pH 9.5 was added to reduce the acetonitrile concentrationto 1%. The trypsin digested HuLys11-IB samples were then transferredinto tubes containing immobilized soybean trypsin inhibitor gel with100× binding capacity to bind the trypsin, rotated to mix for 15 minutesat RT, centrifuged @ 2000 rpm for 2 minutes. The trypsin-depletedHuLys11-IB preparation was then transferred to a tube containing 0.1 mlof Streptavidin-Agarose gel with 2 mg Streptavidin (Say) per ml gel thathad been equilibrated with 50 mM ammonium bicarbonate, pH 9.5, incubatedwith mixing for 1 hour at RT, and centrifuged 2000 rpm for 2 minutes.The HuLys11-IB fragments bound to Sav-agarose were then washedconsecutively with 1 ml per wash of 50 mM ammonium bicarbonate pH9.5+0.2% NP40+2 mM EDTA+150 mM NaCl, then with 50 mM ammoniumbicarbonate pH 9.5+0.2% NP40+2 mM EDTA+500 mM NaCl, and 5 times with 5mM ammonium bicarbonate pH 9.5 to rid the SAv-agarose of nonspecificallybound proteins. The affinity purified HuLys11-IB fragments were theneluted by adding 5 mM citrate buffer, pH 2, rotated for 10 minutes atRT, and centrifuged 2000 rpm for 2 minutes.

The supernatant containing HuLys11-IB fragments were then submitted tothe University of Massachusetts Medical School Mass SpectrometryFacility(http://www.umassmed.edu/proteomic/index.aspx?linkidentifier=id&itemid=10552).Purified samples were analyzed by MALDI-TOF/TOF to identify animinobiotin-derivatized tryptic fragments by both MS data from peptidemixtures as well as from Collisional-Induced-Dissociation (CID) analysisof individual peptides. The Mascot (www.matrixscience.com) searchalgorithm generated several masses generated two masses (MR 2384 and1491) that mapped to the HuLys11 heavy sequence. The sequence,FNWYVDGVEVHNAKTKPR (SEQ ID NO: 1), had an iminobiotin linkage to theposition 288 lysine residue (underlined) and the second sequence,VSNKALPAPIEK (SEQ ID NO: 3), had its only lysine (underlined) inposition 326 conjugated in the same manner. DNA sequence analysis of theHuLys11 clone and translation into the amino acid resulted in thefollowing sequence:

(SEQ ID NO: 18) THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

Alignment of the peptide sequences (SEQ ID NO: 1 and SEQ ID NO: 3) tothe Fc fragment of the HuLys11 amino acid sequence (SEQ ID NO: 18)reveal that both candidate lysine residues map to the Fc fragment andmaking them candidates for the increased plasma half-life. The firstlysine residue is positioned lysine 288 and the second is positionedlysine 326, both based on amino acid nomenclature used in theapplication for the Fc domain is according to the EU index of Kabat etal., “Sequences of Proteins of Immunological Interest”, 5th ed.,National Institutes of Health, Bethesda, Md. (1991).

Example 15: Identification of Critical Lysine Residues for Biotinylation

To establish a direct cause and effect between candidate biotinylatedlysine residues 288 and 326 of hIgG1 and an increase in plasma half-lifein vivo, it is critical to functionally evaluate each candidate's rolein extending the half-life of hIgG. To do so, two types of site-specificmutagenesis of the Fc gene candidate lysine codons can be performed: (1)elimination of the lysines; and (2) site-specific mutagenesis such thatonly one lysine residue is available for biotinylation.

(1) Elimination of candidate lysines. We have PCR cloned and sequencevalidated the HuLys11 heavy and light chains, which we have cloned intopcDNA 3.3-TOPO (heavy chain) and pOptiVEC-TOPO (light chain) vectors.Oligonucleotide-based mutagenesis can be employed to convert thecandidate lysine residues 288 and 326 described above to arginine (R) bysite specific mutagenesis using standard techniques known in the art.Arginine was chosen because it is similar in charge and size to lysine,but cannot be derivatized with NHS-biotin because it lacks an epsilonamine. The mutated heavy chains in the pcDNA 3.3-TOPO mammalianexpression vector can then be transfected into CHO cells in combinationwith the pOptiVEC-TOPO (light chain) vector. Mutant and wild typeHuLys11 antibodies isolated from the transfected CHO culture supernatantcan then be protein G column purified.

To determine whether the lysine to arginine-substituted amino acidsalter the in vivo half-life of HuLys11 antibodies, purified, mutant andwild type control HuLys11 antibodies can be conjugated with NHS-biotinor left unconjugated, and then administered to mFcRn−/−hFcRn transgenicmice. Serial serum samples can be analyzed by ELISA using hen egglysozyme (HEL) as the capture ligand. Reduction in the half-life ofNHS-biotinylated mutant variants as compared with the WT HuLys11 biotinconjugate would confirm the importance of the relevant lysine residue ascausal to the biotin effect. Parallel experiments can be performed usingthe transplacental transfer assay described in Example 16. Furthermore,the same modified proteins can be evaluated for binding human FcRn invitro as described in Example 13 to establish whether changes inhalf-life in vivo and avidity in vitro correlate.

Example 16: Determination if Biotinylation Improves the TherapeuticEfficacy of IVIg

Therapeutics benefits of high doses (1 g/kg) of IVIg have beenattributed to the ability of IVIg to saturate the FcRn recyclingpathway, resulting in the increased catabolic elimination of pathogenicendogenous antibodies (Curr Opin Immunol. 2007 December; 19(6):646-51.Epub 2007 Nov. 26; J Clin Invest. 2005 December; 115(12):3440-50. Epub2005 Nov. 10; J Clin Invest. 2004 May; 113(9):1328-33; J Pharm Sci. 2007June; 96(6):1625-37. Commercially available GammaGard® (Baxter Labs) areconjugated with NHS-biotin, or unconjugated, after its hIgG fraction hasbeen isolated by Protein G chromatography. Doses of NHS-biotinylatedIVIg and unconjugated IVIg ranging from 1 (expected subsaturation) to 5mg/G (expected saturation based on previous studies) body weight arethen injected once into groups of 5 C57BL/6 mFcRn−/−hFcRn+/Tg Line 276mice that have previously received a tracer dose of the anti-HEL HuLys11hIgG1. Serum concentrations of the anti-HEL HuLys11 mAb from serialblood samples can be assayed by ELISA, and serum half lives determined.This experiment tests whether biotinylated IVIg is more efficient atpromoting the clearance of the anti-HEL HuLys11 mAb tracer thanunbiotinylated IVIg.

As a model for arthritis, the human arthritis serum transfer modeldescribed in Petkova et al 2006 (Petkova et al., 2006c) can be employed.In this model, serum (or purified IgG) from certain patients withrheumatoid arthritis caused transient arthritic episodes upon transferinto the Fcgr2b−/−mouse model. In this model, IVIg ameliorates theinflammatory effects of arthritic human serum when administered insaturating doses into Fcgr2b−/−mFcRn−/−hFcRn Tg 276 mice (FIG. 13).Groups of Fcgr2b−/−mFcRn−/−hFcRn Tg 276 mice treated with humanarthritic serum from a preexisting pool of serum known to evoke robustankle lesions are also administered limiting doses of biotinylated andunmodified control IVIg extrapolated from the above-described dosingexperiment. Prior to treatment, the mice are injected with HuLys11 hIgG1tracer, making it possible to monitor the overall effect of treatment onthe serum hIgG half life. The mice are monitored for ankle width and forarthritis score (a combination of redness, swelling and overallinflammation) (Korganow et al., 1999; Petkova et al., 2006b). Thisexperiment tests whether biotinylated IVIg more effectively confersanti-inflammatory effects compared with unbiotinylated IVIg.

Example 17: Determination if Biotinylation Improves TransplacentalTransport of hIgG

The transplacental transport assay can be used to measure transcytosisof hIgG across the fetal trophoblast and the vascular endothelium(Al-Khabbaz, 2008). Pregnant C57BL/6 mFcRn−/−hFcRn+/Tg Line 276 mice areinjected with 500 μg/mouse of biotinylated and un-conjugated hIgGdiluted in 1×PBS to 200 μl/mouse total volume injected intraperitoneallyat day 17 of gestation. Blood samples are collected from gestation stageday 19 and fetuses are collected. The level of biotinylated andunconjugated hIgG in the serum of both the mother and fetus can bedetermined by a standard sandwich ELISA.

Example 18: Determination of Whether Biotinylation is Additive withOther Types of Modifications Known to Enhance Half-Lives andTranscytosis

It is known in the art that the substitution of certain amino acids(e.g. the Hu4D5-IgG1 N434A) substantially increase the half-life; forexample in human FcRn transgenic mice and non-human primates in(Dall'Acqua et al., 2006; Hinton et al., 2007; Hinton et al., 2006;Petkova et al., 2006a). Furthermore, it is known that such engineeredantibodies can demonstrate enhanced transcytosis (Al-Khabbaz, 2008). Totest if biotinylation results in synergistic or additive effects incombination with these defined amino acid modifications, variantantibodies with alanine replacements in key amino acids (e.g., IgG1N434A) are biotinylated, as described in Example 1. To evaluate plasmahalf-lives in vivo, 100 μg of the biotinylated and un-conjugated variantantibody are compared. 100 μg of each antibody can be injectedintraperitoneally into C57/BL6 mFcRn−/−hFcRn+/Tg Line 276 mice (5mice/group) at day −3. Plasma can be collected serially from mice andthe plasma concentrations of the injected antibodies are measured byELISA as described in Example 2. The finding that the biotinylatedversion may show longer plasma half-life compared with thenon-biotinylated version would be consistent with synergistic/additiveeffects. The same preparations can also be analyzed for transcytosisusing the transplacental assay (see FIG. 15) to determine whetherbiotinylation acts synergistically with amino acid substitutions knownto enhance transport.

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INCORPORATION BY REFERENCE

All publications and patents mentioned herein are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually indicated to be incorporated byreference.

While specific embodiments of the subject invention have been discussed,the above specification is illustrative and not restrictive. Manyvariations of the invention will become apparent to those skilled in theart upon review of this specification and the claims below. The fullscope of the invention should be determined by reference to the claims,along with their full scope of equivalents, and the specification, alongwith such variations.

We claim:
 1. A method of treating a disorder that can be treated byintravenous immunoglobulin (IVIg) therapy in a subject in need thereof,comprising administering to the subject an effective amount of anantibody preparation comprising an antibody molecule comprising a humanFc-domain and effective to treat the disorder, wherein the antibody isconjugated to a biotin moiety, wherein the biotin moiety is conjugatedto the antibody molecule in an amount sufficient to increase thehalf-life of the antibody molecule in vivo relative to the half-life ofa corresponding antibody molecule that does not comprise a biotin moietyand wherein the biotin moiety is covalently conjugated to a solventexposed lysine within the human Fc domain sequences FNWYVDGVEVHNAKTKPR(SEQ ID NO: 1), FKWYVDGVEVHNAKTKPR (SEQ ID NO: 2), or VSNKALPAPIEK (SEQID NO: 3).
 2. The method of claim 1, wherein the antibody preparationcomprises a monoclonal antibody preparation.
 3. The method of claim 1,wherein the antibody preparation comprises a polyclonal antibodypreparation.
 4. The method of claim 1, wherein the polyclonal antibodypreparation consists essentially of a polyclonal population ofantibodies specific for a single target molecule.
 5. The method of claim1, wherein the polyclonal antibody preparation comprises a polyclonalpopulation of antibodies specific for a plurality of differentmolecules.
 6. The method of claim 1, wherein the polyclonal antibodypreparation is a pooled preparation isolated from a plurality of humans.7. The method of claim 1, wherein the antibody molecule is a humanimmunoglobulin G (IgG) molecule.
 8. The method of claim 1, wherein theamount of biotin moiety conjugation to the antibody is at a ratio of atleast 2 biotin moiety molecules per antibody molecule.
 9. The method ofclaim 1, wherein the subject is a human.